Anti-TSG101 antibodies and their uses for treatment of viral infections

ABSTRACT

The present invention provides antibodies that bind to the C-terminal region of TSG101. The invention also provides methods of using the TSG101 antibodies for the treatment of viral infections, including HIV and Ebola virus infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit, under 35 U.S.C. §119(e), of U.S.Provisional Patent Application No. 60/858,922, filed on Nov. 15, 2006,which is hereby incorporated by reference in its entirety. Thisapplication is related to U.S. patent application Ser. No. 11/939,122filed Nov. 13, 2007, also incorporated herein-by-reference. U.S. Ser.No. 11/939,122 is directed to the preparation and use, inter alia, ofantibodies directed to a family of proteins that are implicated in viralbudding, referred to therein as escort proteins. TSG101 is one member ofthat family. It has a significance all its own.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to antibodies that bind a TSG101 proteinand inhibit or reduce viral production. The invention also relates tomethods of using the TSG101 antibodies for the treatment of viralinfections, including HIV infection. The invention further relates tomethods of detecting viral infected cells using TSG101 antibodies.

2. Background of the Technology

Pathogen and host cell interactions play critical roles in thepathogenesis of viral diseases such as AIDS. For a typical viralinfection, viruses have to attach to the host cells through cell surfacereceptors, fuse with host cell membrane, translocate across the cellmembrane, uncoat viral particles, synthesize and assemble viral proteinsusing host protein synthesis machinery, and release from host cellsthrough host exporting machinery. The interplay between the viruses andhost cells determine the outcome of viral pathogenesis, ranging from theelimination of viruses to a parasitic or lethal infection. For example,HIV employs a variety of strategies to productively infect human cells.A retrovirus, its life cycle begins by attaching to host cells- theprimary target is the CD4+ T helper cells and gaining entry via specificreceptors. In the cell, the RNA genome is “reverse” transcribed to itscomplementary DNA, and then shuttled to the nucleus for its integrationin the host genome. This integrated “provirus” then directs theproduction of new viral RNA and proteins, which self-assemble and then“bud” from the cell as mature- and infectious-viral particles, envelopedin plasma membrane. Like all viruses, the HIV is a parasite, unable tocatalyze the membrane fission event that drives the budding process.Instead, the nascent virus recruits the cell's membrane sortingmachinery to complete this final stage of infection. Such an host andvirus interplay has been well demonstrated in individuals, who carry adefective cell surface receptor (CCR5), are completely resistant to HIVinfection, elucidating the important roles of host genes and geneticpathways in viral pathogenesis.

Tumor Susceptibility Gene 101 (TSG101, Li, et al., 1996, Cell 85,319-29) plays important roles in cell growth (Zhong, et al., 1998,Cancer Res. 58, 2699-702; Oh, et al., 2002, Proc. Natl. Acad. Sci. USA99, 5430-5; Krempler, et al., 2002, J. Biol. Chem. 277, 43216-23;Wagner, et al., 2003, Mol. Cell. Biol. 23, 150-62; Li, et al., 1996,Cell 85, 319-29), cellular protein trafficking (Babst, et al., 2000,Traffic 1, 248-58; Bishop, et al., 2002, J. Cell Biol. 157, 91-101), anddegradation of p53 (Li, et al., 2001, Proc. Natl. Acad. Sci. USA 98,1619-24; Ruland, et al., 2001, Proc. Natl. Acad. Sci. USA 98, 1859-64;Moyret-Lalle, et al., 2001, Cancer Res. 61, 486-8). TSG101 is alsowidely recognized as a key player in this final stage, inhibition ofcellular TSG101 blocks the budding process of HIV. Acting in concertwith other cellular factors, TSG 101 thus plays an essential role in thebudding or spread of HIV viruses. The HIV Gag protein, previously shownto orchestrate viral assembly and budding, binds with high affinity toTSG 101, and this Gag/TSG101 interaction is essential for efficient HIVviral assembling and budding, as disruption of the Gag/TSG101interaction prevents HIV viral budding, and significantly limit thespread of HIV virus.

The final step in the assembly of an enveloped virus assembly requiresseparation of budding particles from the cellular membranes. Threedistinct functional domains in Gag,20, i.e., PTAP in HIV-1 [SEQ ID NO:44](Gottlinger, et al., 1991, Proc. Natl. Acad. Sci. USA 88, 3195-9;Huang, et al., 1995, J. Virol. 69, 6810-8); PPPY in RSV [SEQ ID NO:45](Parent, et al., 1995, J. Virol. 69, 5455-60), MuLV (Yuan, et al.,1999, Embo. J. 18, 4700-10), and M-PMV (Yasuda, et al., 1998, J. Virol.72, 4095-103); and YXXL in EIAV (Puffer, et al., 1997, J. Virol. 71,6541-6), have been identified in different retroviruses that arerequired for this function and have been termed late, or L domains(Wills, et al., 1991, Aids 5, 639-54). In HIV-1, the L domain contains aPTAP motif and is required for efficient HIV-1 release (see, e.g.,Wills, et al., 1994, J. Virol. 68, 6605-6618; Gottlinger, et al., 1991,Proc. Natl. Acad. Sci. USA 88, 3195- 3199; Huang, et al., 1995, J.Virol. 69, 6810-6818). The L domain of HIV-1 p6, especially the PTAPmotif, binds to the cellular protein TSG101 and recruits it to the siteof virus assembly to promote virus budding (VerPlank, et al., 2001,Proc. Natl. Acad. Sci. USA, 98:7724-7729; Garrus, et al., 2001, Cell107:55-65; Martin-Serrano, et al., 2001, Nature Medicine 7:1313-19;Pornillos, et al., 2002, EMBO J. 21:2397-2406; Demirov, et al., 2002,Proc. Natl. Acad. Sci. USA 99:955-960; PCT Publication WO 02/072790;U.S. Patent Application Publication No. US 2002/0177207). The UEV domainof TSG101 binds the PTAP motif and mono-ubiquitin (Pornillos, et al.,2002, Embo J. 21, 2397-406; Pornillos, et al., 2002, Nat. Struct. Biol.9, 812-7), which has also been implicated in HIV-1 budding (Patnaik, etal., 2000, Proc. Natl. Acad. Sci. USA 97, 13069-74; Schubert, et al.,2000, Proc. Natl. Acad. Sci. USA 97, 13057-62; Strack, et al., 2000,Proc. Natl. Acad. Sci. USA 97, 13063-8). Depletion of cellular TSG101(Garrus, et al., 2001, Cell 107:55-65) or over-expression of a truncatedform of TSG101 inhibits HIV-1 release (Demirov, et al., 2002, Proc.Natl. Acad. Sci. USA 99:955-960). Under certain circumstances, TSG101can even substitute for the HIV-1 L domain to promote virus release(Martin-Serrano, et. al., 2001, Nature Medicine 7:1313-19).

In yeast, the Tsg101 ortholog Vps23 has been shown to interact withVps28 and Vps37 and to form a protein complex named ESCRT-I, which iscritical for endosomal protein sorting into the multivesicular bodypathway (Katzmann, et al., 2001, Cell 106, 145-55). It is hypothesizedthat this intracellular multivesicular body formation resembles HIV-1release at the plasma membrane (Garrus, et al., 2001, Cell 107:55-65;Patnaik, et al., 2000, Proc. Natl. Acad. Sci. USA 97, 13069-74). Inmammalian cells, TSG101 interacts with Vps28 to form an ESCRT-I-likecomplex (Babst, et al., 2000, Traffic 1, 248-58; Bishop, et al., 2002,J. Cell Biol. 157, 91-101; Bishop, et al., 2001, J. Biol. Chem. 276,11735-42), although the mammalian homolog of Vps37 has not beenidentified.

Recent studies (Blower, et al., 2003, AIDS Rev. 5:113-25; Valdiserri, etal., 2003, Nat. Med. 9:881-6) have estimated that as many as 42 millionpeople worldwide have been infected with HIV. The disease has killedmore than 3 million people. While the advent of highly potent andtargeted combination therapies has slowed the progression of AIDS inindustrialized nations, the AIDS pandemic is causing a “humandevelopment catastrophe” in developing nations, particularly in Africa,where more than 21 million Africans have been infected. In South Africaalone, the death toll is projected to rise to 10 million by 2015.Related statistics portend a similar crisis in the Asia Pacific region,which, according to United Nations' estimates, has more than 7 millionHIV-infected individuals. Repercussions from the AIDS pandemic extendwell beyond the clinic, which lack the resources to treat the swellingnumbers of recently infected patients (nearly 20% of the adultpopulation in South Africa is infected). Treatment of HIV-infected andgravely ill AIDS patients is stressing the already over-burdened healthcare systems of Africa and other developing nations. Worse yet, currenttreatments for HIV

despite their initial success in reducing viral load

are beginning to lose their efficacy, as drug-resistant HIV strains areincreasingly isolated in newly infected individuals. Further compoundingthe therapeutic management of HIV disease is the toxicity of currentantiretroviral regimens, the magnitude of which complicates thephysician's decision to begin and to maintain treatment. Identifying newtherapeutic paradigms for the treatment of HIV disease, especially thosewith mechanisms of action that promise to slow the development ofresistance, is indeed a global challenge for the pharmaceuticalindustry.

Many viruses are also highly mutable. Methods and compositions relyingon targeting such viruses directly are normally not sufficient in thetreatment of infection by such viruses. For example, HIV-1 is such ahighly mutable virus that during the course of HIV-1 infection, theantibodies generated in an infected individual do not provide permanentprotective effect due in part to the rapid emergence of neutralizationescape variants (Thali, et al., 1992, J. Acquired Immune DeficiencySyndromes 5:591-599). Current therapies for the treatment ofHIV-infected individuals focus primarily on viral enzymes involved intwo distinct stages of HIV infection, the replication of the viralgenome and the maturation of viral proteins. Since the virus frequentlymutates, strains resistant to an antiviral inhibitor develop quickly,despite the drug's initial therapeutic effects. In one recent study, thepercentage of individuals newly infected with drug-resistant HIV strainsincreased six fold over a five year period (Little, et al., 2002, N.Engl. J. Med. 347:385-94). Further, combination therapy, the currentstandard of care that attacks HIV with inhibitors of both reversetranscriptase and protease, is leading to the development of multi-drugresistant HIV strains. Anti-retroviral drugs directed against newHIV-based targets, while of considerable value, do not address thisincreasingly critical issue. For example, HIV strains resistant toFuzeon® (enfuvirtide), the newest addition to the anti-HIVarmamentarium, have already been isolated from patients. Thus, despiteits antiviral potency and novel mechanism of action, drug-resistance islikely to undermine the therapeutic potential of viral fusion inhibitorslike Fuzeon®. There is therefore a need for developing noveltherapeutics and preventative measures to combat viral infections suchas HIV infection.

Discussion or citation of a reference herein shall not be construed asan admission that such reference is prior art to the present invention.

SUMMARY OF THE INVENTION

The present invention provides antibodies that binds to the terminalregions of human TSG101 and methods of using the antibodies for treatingviral infections. The present invention also provides methods andcompositions for treating viral infection using the anti-TSG101antibodies.

In one aspect, the present invention provides monoclonal antibodies thatbinds to the TSG101 C-terminal region consisting of SEQ ID NO:3. In oneembodiment, the antibody is a monoclonal antibody comprising apolypeptide comprising an amino acid sequence recited in SEQ ID NO: 19,22, 25 or 28.

In another embodiment, the monoclonal antibodies are antibody D1 and3G1.

In another embodiment, the antibody is a humanized antibody comprisingan amino acid sequence selected from the group consisting of SEQ IDNOS:30-41.

In a preferred embodiment, the humanized antibody comprises either theamino acid sequences recited in SEQ ID NOS:31-35 or the amino acidsequences recited in SEQ ID NOS:36-41.

Another aspect of the present invention relates to a method for treatingan enveloped virus infection in a subject. The method comprisesadministering into the subject an effective amount of the anti-TSG101antibody of the present invention.

In one embodiment, the enveloped virus is a human immunodeficiencyvirus, Marburg virus, or Ebola virus.

Yet another aspect of the present invention relates to a pharmaceuticalcomposition for treating human immunodeficiency virus, Marburg virus,and Ebola virus infections. The composition comprises an effectiveamount of the anti-TSG101 antibody of the present invention; and apharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the 390 amino acid sequence of human TSG101 protein (SEQID NO:1). (GenBank Accession No. U82130.1/GI:1772663).

FIG. 2 depicts the effects of anti-TSG101 antibodies on MLV virusproduction. Left top panel: phoenix helper cells without treatment ofantibody (positive control) showed efficient production of retroviruses,and infection of N2A target cells; left middle panel: Rabbit IgG had noeffect; left bottom panel: a rabbit antibody against N-terminal TSG101had an effect of less than 20% inhibition; right top panel: a rabbitantibody against C-terminal TSG101 significantly inhibited theproduction of retroviruses, and infection of

N2A target cells (50% to 70% inhibition); right middle panel: a mixtureof anti-C terminal and anti-N terminal antibodies gave similar resultsas the anti-C terminal antibody alone; right bottom panel: N2a cellsthat were not infected by viruses only showed minimal backgroundstaining.

FIGS. 3A-E show that GFP-TSG101 localizes to cell surface during viralrelease. Live confocal images of Phoenix helper cells with active viralrelease 24 hours after transfection of GFP-TSG101. FIG. 3A shows brightfield images of four cells; FIGS. 3B-E are live confocal fluorescenceimages of the same field at different sections; White arrows point tocell surface localization of GFP-TSG101.

FIG. 4 shows cell surface localization of TSG101 during HIV budding.H9ABg1 cells (CD4+ human T lymphocytes, carrying HIV viral integration)were actively producing and releasing HIV virions with a defectiveenvelope protein (this non-infectious form of HIV viruses will notinfection other cells, thus specifically allowing the study viralrelease). The parental H9 cells that do not carry HIV were used as acontrol. Both H9ABg1 and H9 cells were fixed with 2% paraformoldehydefor 10 min. at room temperature (this surface fixation does notpermeabilize cells). Anti-TSG101 antibody were incubated with both celllines for 20 min. and detected with a fluorescence labeled secondaryantibody. Top panels: fluorescence images; Bottom panels: bright fieldimages.

FIG. 5 shows FACS profile of cell surface localization of TSG101 duringHIV budding. Both H9ΔBg1 and H9 cells were fixed with 2%paraformoldehyde for 10 min. at room temperature (this surface fixationdoesn't permeabilize cells). Anti-TSG101 antibodies were incubated withboth cell lines for 20 min. and detected with a fluorescent labeledsecondary antibody. The immuno-stained cells were analyzed via FACS. Toppanel: H9ΔBg1 cells, with 85% cells stained positive for surface TSG101;Bottom panel: H9 control cells, with less than 0.1% cells stainedpositive for surface TSG101.

FIG. 6 shows inhibition of HIV-1 production by anti-TSG101 antibodies.Lane 1 and 5, mock transfection; Lane 2 and 6, pNL4-3 and controlantibody (rabbit IgG); Lane 3 and 7, pNL4-3 and anti-TSG101 antibody“B”; Lane 4 and 8, pNL4-3 and anti-TSG101 antibody “E”.

FIG. 7 shows antibody inhibition of HIV release from H9ΔBg1 cells. HIVproducing H9ΔBg1 cells were incubated with anti-TSGI01 antibody “E” atdifferent concentrations, 48 hours later, viral supernatants werecollected and assayed by HIV p24 ELISA kit. Averages of threeindependent experiments (each with triplicates) were shown. Significantantibody inhibition (*P<0.05) of viral release was observed at 80 ug/ml.

FIG. 8 shows antibody inhibition of HIV infectivity. HIV producingJurkat cells (infected with Wild-type HIV-1) were incubated withanti-TSG101 antibody “E” at 40 ug/ml.

FIGS. 9A-B show release of Ebola GP and VP40 into culture supernatants.FIG. 9A shows 293T cells were transfected with the indicated plasmids,supernatants were cleared from floating and particulate material werepelleted through 20% sucrose by ultracentrifugation. The individualproteins were detected in the cell lysates and in the particulatematerial from supernatant by immunoblotting (IB). FIG. 9B showssupernatants from cells transfected with Ebola VP40 alone or GP+VP40were immunoprecipitated with anti-GP mAb and analyzed by immunoblotting.Lower panel shows the expression of VP40 in total cell lysates. IgH:immunoglobulin heavy chain from the antibody used forimmunoprecipitation.

FIGS. 10A-B show electron microscopic analysis of virus like particlesgenerated by EBOV GP and VP40. Particles obtained by ultracentrifugationof the supernatants of 293T cells transfected with Ebola GP+VP40 werenegatively stained with uranyl-acetate to reveal the ultrastructure.(FIG. 10A) or stained with anti-Ebo-GP mAb followed by Immunogold rabbitanti mouse Ab (FIG. 10B), and analyzed by electron microscopy.

FIG. 11 shows association of VP40 and TSG101 in 293T cells. Cells weretransfected with Myc tagged TSG101 full length (FL) or the indicatedtruncations along with VP40. After 48 h cells were lysed and subjectedto immunoprecipitatiopn with anti Myc.

FIG. 12 shows Far-Western analysis of association between Ebola VP40 andTSG101 UEV domain.

FIG. 13 shows SPR analysis of Ebola VP40 interaction with TSG101.

FIGS. 14A-B show association of TSG101 with Ebola VLPs and inactivatedEbola virus. In FIG. 14, 293 cells were transfected with the indicatedplasmids, supernatants were immunoprecipitated with anti-GP mAb andanalyzed by immunoblotting with the antibodies indicated on the right.Lower three panel shows the expression of the transfected proteins intotal cell lysates. In FIG. 14B, 5 μg inactivated Ebola virus (iEBOV)were subjected to SDS-PAGE and Western blot analysis with rabbitanti-TSG101 antibody. The molecular weight markers and position ofTSG101 are indicated.

FIG. 15 shows results of inhibition of Ebola virus release in Hela cellsby antiTSG101 antibodies.

FIG. 16 is a graph showing the anti-HIV activity of monoclonal antibodypool PE-8 in the form of purified IgG from ascites.

FIG. 17 is a graph showing the anti-HIV activity of representativesubclones of monoclonal antibody pool PE-8 in the form of hybridomasupernatants.

FIG. 18 is a graph showing the anti-HIV activity of one of the subclonesof monoclonal antibody pool PE-8 in the form of purified IgG fromascites.

FIG. 19 is a graph showing inhibition of drug-resistant HIV strains byTSG101 dominant negative mutant.

FIG. 20 is a graph showing inhibition of drug-resistant HIV strainpL-10R by monoclonal antibody pool PE-8.

FIG. 21 is a graph showing inhibition of drug-resistant HIV strainp1617-1 by monoclonal antibody pool PE-8.

FIGS. 22A and 22B are graphs showing inhibition of HIV production inperipheral blood mononuclear cells at day 3 and day 7 post infection,respectively.

FIG. 23 is a graph showing that anti-TSG101 mabs protect mice againstEBOV challenge.

FIG. 24 is a partial listing of viruses identified by researches asdependent or involving some aspect of TSG101 by other researchers in thefield.

FIG. 25 is a schematic illustration of the preparation of humananti-TSG101 antibodies according to the invention.

FIG. 26 graphically reflects the binding of phage candidates to infectscells detected by immunofluorescence.

FIG. 27 graphically reflects the binding, by the same TSG101 antibody,to different cell types infected with different viruses.

FIG. 28 demonstrates the binding, by staining, of TSG101 antibodies toRSV infected cells, and the absence of binding to controls.

FIG. 29 reflects, in bar graph format, the ability of the TSG101antibodies of the invention to kill infected cells through ADCC.

FIG. 30 reflects, in bar graph format, the ability of the TSG101antibodies of the invention to kill virally infected cells and therebydirectly inhibit viral infection independent of innate host defensemechanisms

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of using antibodies that bind aTSG101 protein to inhibit or reduce viral production. The presentinvention also provides methods of using TSG101 antibodies for treatingviral infections. The present invention also provides methods andcompositions for treating viral infection by targeting TSG101 on thesurface of infected cells, e.g., delivering therapeutic and/ordiagnostic agents, to such infected cells.

The rational design of therapeutics requires an improved understandingof HIV pathogenesis. Recent studies show that a host protein, TSG101,plays a critical role in the pathogenesis of various viruses such as HIVand Ebola viruses. In particular, TSG101 participates in the process bywhich viral particles escape, or bud, from infected cells, and thereforerepresents a novel target for anti-viral drug discovery. Underlying theassembly and release of enveloped RNA viruses from infected cells is atight coordination between viral and host proteins (Perez, et al., 2001,Immunity 15(5): 687-90; Freed, 2002, J. Virol. 76(10): 4679-87;Pornillos, et al., 2002, Trends Cell Biol. 12(12): 569-79). While manyof the protein: protein and protein: membrane interactions that governthe final stages of infection have yet to be identified, the cellularTSG101 protein has emerged as a critical player (Garrus, et al., 2001,Cell 107:55-65; Carter 2002; Pornillos, et al., 2002, Trends Cell Biol.12 (12): 569-79; Pornillos, et al., 2002, Nat. Struct. Biol. 9, 812-7).Genetic, biochemical and microscopic analyses have shown that TSG101interacts with multiple enveloped RNA viruses including members of theretrovirus, rhabdovirus and filovirus families.

Despite their considerable evolutionary divergence, many enveloped RNAviruses employ similar strategies to complete the final stages ofinfection (Martin-Serrano, et. al., 2001, Nature Medicine 7:1313-19;Freed, 2002, J. Virol. 76(10): 4679-87). Of particular importance toHuman Immunodeficiency Virus Type 1 (HIV-1), Vesicular Stomatitis Virus(VSV), Ebola Virus (EBOV), Marburg Virus (MARV) and others is the Lateor L domain, a sequence motif that uniquely appropriates cellularpathways to drive viral particle assembly and budding. Three sequencemotifs with L-domain activity have been characterized: PPxY, YxxL andPTAP (where “x” denotes any amino acid). HIV-1 budding requires the PTAPmotif, found at the amino terminus of the p6Gag protein. Rhabdoviruses,as typified by VSV, utilize the PPxY motif within the Matrix (M)protein. The L-domains of multiple viral families recruit TSG101, acellular protein critical to endosomal membrane sorting (VerPlank, etal., 2001, Proc. Natl. Acad. Sci. USA 98:7724-7729; Pornillos, et al.,2002, Nat. Struct. Biol. 9, 812-7). Initially identified by a randomknockout screen in mammalian cells, TSG101 is a 43 KDa multifunctionalprotein involved in membrane trafficking, cell cycle control,microtubule assembly and protein degradation (Li, et al., 1996, Cell 85,319-29; Bishop, et al., 2001, J. Biol. Chem. 276: 11735-42; Katzmann, etal., 2001, Cell 106, 145-55; Li, et al., 2001, Proc. Natl. Acad. Sci.USA 98, 1619-24. The C-terminus of TSG101 possesses a coiled-coil domainand a domain that auto-regulates its cellular levels; whereas the TSG101amino-terminus

which interacts with multiple viral L-domains via a binding pocket thatstructurally and functionally resembles WW and SH3 domains

bears significant homology to Ubiquitin Conjugating (UBC) E2 enzymes(Pornillos, et al., 2002, Nat. Struct. Biol. 9, 812-7). Although theUBC-like domain of TSG101 strongly binds ubiquitin, a 76 amino acidprotein central to regulating protein turnover and sorting, it lacks thecatalytic cysteine residue involved in ubiquitination of target proteins(Hicke, 2001, Cell 106, 527-30).

In eukaryotic cells, TSG101 is a component of ESCRTI (endosomal sortingcomplex required for transport), a −350 kDa cytoplasmic complex thatalso includes Vps28 and Vps37 (Katzmann, et al., 2001, Cell 106, 145-55;Bishop, et al., 2002, J. Cell Biol. 157, 91-101). The interaction amongthese three proteins, and their respective roles in membrane traffickingare currently under investigation. The TSG101 yeast homologue, Vps23,was identified by its functional complementation of protein sortingdefects (Babst, et al., 2000, Traffic 1, 248-58). Fibroblasts withreduced TSG101 levels and yeast Vps23 null 20 mutants both displaydefects in the endosomal/MVB pathway. For instance, receptors that wouldnormally enter the MVB system for lysosomal degradation are insteadrecycled to the surface, leading to profound disturbances in cellsignaling. Based on their recent experimental analysis, Katzmann, et al.have suggested that TSG101Nps23 binds ubiquitinated proteins at thesurface of early endosomes, and facilitates their entry into MVBvesicles (Katzmann, et al., 2001, Cell 106, 145-55).

In addition to TSG101, cellular proteins with WW-domains have been shownto interact with the L-domain sequence motifs of enveloped RNA viruses(Harty, et al., 2000, Proc. Natl. Acad. Sci. USA 97, 13871-6; Kikonyogo,et al., 2001, Proc. Natl. Acad. Sci. USA 98, 11199-204). For example,Far-Western binding assays have demonstrated a specific interaction withthe WW-domains of the mammalian ubiquitin ligase, Nedd4, and its yeasthomolog Rsp5, with the VP40 L domain of EBOV (Harty, et al., 2000, Proc.Natl. Acad. Sci. USA 97, 13871-6; Kikonyogo, et al., 2001, Proc. Natl.Acad. Sci. USA 98, 11199-204). Indeed, the data thus far point to animportant role for ubiquitin in viral budding (Patnaik, et al., 2000,Proc. Natl. Acad. Sci. USA 97, 13069-74; Carter, 2002, Trends Microbiol.10, 203-5; Myers, et al., 2002, J. Virol. 76, 11226-35). There may alsobe a constitutive interaction between Nedd4 and TSG101. It has beensuggested that HIV-1 may exploit Nedd4 and TSG101 to escape frominfected cells in a manner wholly unrelated to the endosomal/MVBpathway. Nevertheless, TSG101 is widely regarded as a key host factorappropriated by viruses to drive viral release. The proposed TSG101/MVBlink is based, in part, on the biophysical process of MVB formation,which is known to include the invagination of the endosomal lipidbilayer away from the cytoplasm and towards the lumen (Patnaik, et al.,2000, Proc. Natl. Acad. Sci. USA 97, 13069-74; Jasenosky, et al., 2001,J. Virol. 75, 5205-14). Enveloped RNA viruses face similar topologicalparameters: following viral assembly on the inner leaflet of themembrane, the bilayer mustevaginate towards the extracellular milieu

again away from the cytoplasm. Devoid of any catalytic ability to splitan otherwise thermodynamically stable bilayer, viruses apparentlyrecruit endosomal membrane factors for assistance. The TSG101: L domaininteraction may thus provide a vital nexus between nascent virions andthe endosomal machinery that drives membrane fission and budding. Asdiscussed above, TSG101, a constituent of ESCRT-1, sorts ubiquitinatedproteins for inclusion in the MVB pathway. But this sorting may besubverted in cells infected with HIV and related enveloped RNA viruses.That is, rather than directing ubiquitinated proteins into the MVBpathway, TSG101 and its endosomal counterparts may direct the plasmamembrane and its associated viral particles to evaginate, formingenveloped vesicles that pinch off from the plasma membrane.

The molecular determinants that drive virion assembly and release arestill an area of active research, though some general conclusions haveemerged. First, the recruitment of TSG101 to the plasma membrane duringvirion maturation is absolutely required. The data supporting a centralTSG101 role are compelling: (i) overexpression of the TSG101 UBC domaintrans-dominantly disrupts VLP formation in HIV-1 Gag expressing cells(Demirov, et al., 2002, Proc. Natl. Acad. Sci. USA 99, 955-960); (ii)ablating TSG101 expression via RNA interference impairs HIV-1 budding(Garrus, et al., 2001, Cell 107, 55-65) and (iii) in both of theseinstances, electron microscopic analysis demonstrated viral particlestethered to the plasma membrane via membranous stalks, structurallysimilar to those found in cells expressing L-domain defective viruses.As shown by Martin-Serrano, et al., L-domain point mutations thatpreclude TSG101 binding with the filoviral VP40 or HIV-1 p6Gag, markedlyreduce viral particle release from human cells, an effect that coincideswith the failure of TSG101 to colocalize with the viral proteins at thelipid bilayer (Martin-Serrano, et. al., 2001, Nature Medicine 7,1313-19). Related experiments demonstrated that the EBOV L-domain wasable to substitute for the p6Gag L-domain, with no discernible effectson VLP release, underscoring the conserved nature of the enveloped RNAviral budding mechanisms. Significantly, HIV-1 L-domain is dispensableonce TSG101 is directed fused to HIV-1 Gag. Therefore the primaryresponsibility of the L-domain is to recruit TSG101 to the plasmamembrane. This interaction between TSG101 and viral L domains representsa novel target for the prevention and treatment of HIV, EBOV and MARVinfections (Luban, 2001, Nat. Med. 7, 1278-80; Senior, 2001, DrugDiscov. Today 6, 1184-1186).

The inventor has discovered that anti-TSG101 antibodies can be used forinhibiting or reducing viral infections.

Anti-TSG101 Antibodies

The invention encompasses the use of an antibody that contains a bindingsite which specifically binds a TSG101 protein for inhibiting orreducing viral infection. Such anti-TSG101 antibodies can therefore beused as broad spectrum anti-viral agents. The term “antibody” as usedherein refers to immunoglobulin molecules. In one embodiment, theantibody binds a C-terminal region of a TSG101 protein. In a preferredembodiment, the antibody binds an epitope comprised in the amino acidregion QLRALMQKARKTAGLSDLY (SEQ ID NO:3). In another preferredembodiment, the antibody is a monoclonal antibody comprising apolypeptide comprising an amino acid sequence recited in SEQ ID NO: 19,22, 25 or 28. In a more preferred embodiment, the monoclonal antibodiesare antibody D1 and 3G1. In another preferred embodiment, the antibodyis a humanized antibody comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOS:30-41. In a more preferredembodiment, the humanized antibody comprises either the amino acidsequences recited in SEQ ID NOS:31-35 or the amino acid sequencesrecited in SEQ ID NOS:36-41.

In another embodiment, the antibody binds an N-terminal region of aTSG101 protein. In a preferred embodiment, the antibody binds an epitopecomprised in the amino acid region VRETVNVITLYKDLKPVL (SEQ ID NO:2).

As used herein, “epitope” refers to an antigenic determinant, i.e., aregion of a molecule that provokes an immunological response in a hostor is bound by an antibody. This region can but need not compriseconsecutive amino acids. The term epitope is also known in the art as“antigenic determinant.” An epitope may comprise as few as three aminoacids in a spatial conformation which is unique to the immune system ofthe host. Generally, an epitope consists of at least five such aminoacids, and more usually consists of at least 8-10 such amino acids.Methods for determining the spatial conformation of such amino acids areknown in the art.

The invention also envisions the use of antibody fragments that containa binding site which specifically binds a TSG101 protein. Examples ofimmunologically active fragments of immunoglobulin molecules includeF(ab) and F(ab′)2 fragments which can be generated by treating theantibody with an enzyme such as pepsin or papain. Examples of methods ofgenerating and expressing immunologically active fragments of antibodiescan be found in U.S. Pat. No. 5,648,237 which is incorporated herein byreference in its entirety.

The immunoglobulin molecules are encoded by genes which include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regions, aswell as a myriad of immunoglobulin variable regions. Light chains areclassified as either kappa or lambda. Light chains comprise a variablelight (V_(L)) and a constant light (C_(L)) domain. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively.Heavy chains comprise variable heavy (V_(H)), constant heavy 1 (CH1),hinge, constant heavy 2 (CH2), and constant heavy 3 (CH3) domains. TheIgG heavy chains are further sub-classified based on their sequencevariation, and the subclasses are designated IgGI, IgG2, IgG3 and IgG4.

Antibodies can be further broken down into two pairs of a light andheavy domain. The paired V_(L) and V_(H) domains each comprise a seriesof seven subdomains: framework region 1 (FR1), complementaritydetermining region 1 (CDR1), framework region 2 (FR2), complementaritydetermining region 2 (CDR2), framework region 3 (FR3), complementaritydetermining region 3 (CDR3), framework region 4 (FR4) which constitutethe antibody-antigen recognition domain.

A chimeric antibody may be made by splicing the genes from a monoclonalantibody of appropriate antigen specificity together with genes from asecond human antibody of appropriate biologic activity. Moreparticularly, the chimeric antibody may be made by splicing the genesencoding the variable regions of an antibody together with the constantregion genes from a second antibody molecule. This method is used ingenerating a humanized monoclonal antibody wherein the complementaritydetermining regions are mouse, and the framework regions are humanthereby decreasing the likelihood of an immune response in humanpatients treated with the antibody (U.S. Pat. Nos. 4,816,567, 4,816,397,5,693,762; 5,585,089; 5,565,332 and 5,821,337 which are incorporatedherein by reference in their entirety).

An antibody suitable for use in the present invention may be obtainedfrom natural sources or produced by hybridoma, recombinant or chemicalsynthetic methods, including modification of constant region functionsby genetic engineering techniques (U.S. Pat. No. 5,624,821). Theantibody of the present invention may be of any isotype, but ispreferably human IgG1.

Antibodies exist for example, as intact immunoglobulins or can becleaved into a number of well-characterized fragments produced bydigestion with various peptidases, such as papain or pepsin. Pepsindigests an antibody below the disulfide linkages in the hinge region toproduce a F(ab)′2 fragment of the antibody which is a dimer of the Fabcomposed of a light chain joined to a VH-CHI by a disulfide bond. TheF(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the F(ab)′2 dimer to aFab′ monomer.

The Fab′ monomer is essentially an Fab with part of the hinge region.See Paul, ed., 1993, Fundamental Immunology, Third Edition (New York:Raven Press), for a detailed description of epitopes, antibodies andantibody fragments. One of skill in the art will recognize that suchFab′ fragments may be synthesized de novo either chemically or usingrecombinant DNA technology. Thus, as used herein, the term antibodyfragments includes antibody fragments produced by the modification ofwhole antibodies or those synthesized de novo.

As used herein, an antibody can also be a single-chain antibody (scFv),which generally comprises a fusion polypeptide consisting of a variabledomain of a light chain fused via a polypeptide linker to the variabledomain of a heavy chain.

The invention also encompasses the use of a polyclonal population ofanti-TSG101 antibodies for inhibiting or reducing viral infection. Asused herein, a polyclonal population of anti-TSGI01 antibodies of thepresent invention refers to a population of anti-TSG101 antibodies,which comprises a plurality of different anti-TSG101 antibodies eachhaving a different binding specificity. In one embodiment, thepopulation of anti-TSG101 antibodies comprises antibodies that bind aC-terminal region of a TSG101 protein. In a preferred embodiment, thepopulation of anti-TSG101 antibodies comprises antibodies that bind oneor more epitopes comprised in the amino acid region QLRALMQKARKTAGLSDLY(SEQ ID NO:3). In another embodiment, the population of anti-TSG111antibodies comprises antibodies that bind an N-terminal region of aTSG101 protein. In a specific embodiment, the population of anti-TSG101antibodies comprises antibodies that bind one or more epitopes comprisedin the amino acid region VRETVNVITLYKDLKPVL (SEQ ID NO:2).

Preferably, the plurality of anti-TSG101 antibodies of the polyclonalpopulation includes specificities for different epitopes of TSG101protein. In preferred embodiments, at least 90%, 75%, 50%, 20%, 10%, 5%,or 1% of anti-TSG101 antibodies in the polyclonal population target thedesired epitopes. In other preferred embodiments, the proportion of anysingle anti-TSG101 antibody in the polyclonal population does not exceed90%, 50%, or 10% of the population. The polyclonal population comprisesat least 2 different anti-TSG101 antibodies with differentspecificities. More preferably, the polyclonal population comprises atleast 10 different anti-TSG101 antibodies. Most preferably, thepolyclonal population comprises at least 100 different anti-TSG101antibodies with different specificities.

Production of Anti-TSG101 Antibodies

TSG101 protein or a fragment thereof can be used to raise antibodieswhich bind TSG101 protein. Such antibodies include but are not limitedto polyclonal, monoclonal, chimeric, single chain, Fab fragments, and anFab expression library. In a preferred embodiment, anti C-terminalTSG101 antibodies are raised using an appropriate C-terminal fragment ofa TSG101 protein. Such antibodies are useful in inhibiting viralproduction.

Production of Monoclonal Anti-TSG101 Antibodies

Antibodies can be prepared by immunizing a suitable subject with aTSG101 protein or a fragment thereof as an immunogen. The antibody titerin the immunized subject can be monitored over time by standardtechniques, such as with an enzyme linked immunosorbent assay (ELISA)using immobilized polypeptide. If desired, the antibody molecules can beisolated from the mammal (e.g., from the blood) and further purified bywell-known techniques, such as protein A chromatography to obtain theIgG fraction. In one embodiment, a polyclonal anti-N terminal TSG101antibody (also referred to as anti-TSG101 antibody “C”) is raised usingan N-terminal fragment of the human TSG101 protein: VRETVNVITLYKDLKPVL(SEQ ID NO:2). In another embodiment, a polyclonal anti-C terminalTSG101 antibody (also referred to as anti-TSG101 antibody “E”) is raisedusing a C-terminal fragment of the human TSG101 protein:QLRALMQKARKTAGLSDLY (SEQ ID NO:3). In yet another embodiment, monoclonalanti-C terminal TSG101 antibody (e.g., pool PE-8, mab D1, and mab 3G1)are raised using a C-terminal fragment of the human TSG101 protein (SEQID NO:3).

At an appropriate time after immunization, e.g., when the specificantibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975, Nature 256:495-497), the human B cellhybridoma technique by Kozbor, et al. (1983, Immunol. Today 4:72), theEBV-hybridoma technique by Cole, et al. (1985, Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. Thetechnology for producing hybridomas is well known (see Current Protocolsin Immunology, 1994, John Wiley & Sons, Inc., New York, N.Y.). Hybridomacells producing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindthe polypeptide of interest, e.g., using a standard ELISA assay.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies. For example, the monoclonal antibodiesmay be made using the hybridoma method first described by Kohler, etal., 1975, Nature, 256:495, or may be made by recombinant DNA methods(U.S. Pat. No. 4,816,567). The term “monoclonal antibody” as used hereinalso indicates that the antibody is an immunoglobulin.

In the hybridoma method of generating monoclonal antibodies, a mouse orother appropriate host animal, such as a hamster, is immunized ashereinabove described to elicit lymphocytes that produce or are capableof producing antibodies that will specifically bind to the protein usedfor immunization (see, e.g., U.S. Pat. No. 5,914,112, which isincorporated herein by reference in its entirety).

Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA.

Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor,1984, J. Immunol., 133:3001; Brodeur, et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987)). Culture medium in which hybridoma cells are growing isassayed for production of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by hybridoma cells is determined by immunoprecipitation or byan in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immuno-absorbent assay (ELISA). The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson, et al., 1980, Anal. Biochem., 107, 220.

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103,Academic Press, 1986). Suitable culture media for this purpose include,for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cellsmay be grown in vivo as ascites tumors in an animal. The monoclonalantibodies secreted by the subclones are suitably separated from theculture medium, ascites fluid, or serum by conventional immunoglobulinpurification procedures such as, for example, protein A-Sepharose,hydroxylapatite chromatography, gel electrophoresis, dialysis, oraffinity chromatography.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody directed against a TSG101 protein or a fragmentthereof can be identified and isolated by screening a recombinantcombinatorial immunoglobulin library (e.g., an antibody phage displaylibrary) with the TSG101 protein or the fragment. Kits for generatingand screening phage display libraries are commercially available (e.g.,Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene antigen SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, U.S. Pat. Nos. 5,223,409 and 5,514,548; PCT PublicationNo. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO92/09690; PCT Publication No. WO 90/02809; Fuchs, et al., 1991,Bio/Technology 9:1370-1372; Hay, et al., 1992, Hum. Antibod. Hybridomas3, 81-85; Huse, et al., 1989, Science 246, 1275-1281; Griffiths, et al.,1993, EMBO J. 12, 725-734.

Functional Genetics, Inc., assignee of this patent application, ofGaithersburg, Md. derived a panel of antibodies that selectively targetTSG101 on virally-infected cells. In brief summary, a naïve scFv librarywas isolated from an anonymous human donor and was used to generate astandard phage library. TSG101 immunoreactivity was determined byimmobilizing purified, full-length TSG101, or polypeptides encompassingdifferent regions of TSG101 and then isolating phage that boundimmobilized TSG101 using an ELISA-based format in situ.

As shown in FIG. 25, after multiple rounds of screening, the resultingphage-based scFv candidates were screened for their abilities to bindTSG101 that is uniquely exposed on the surface of virally-infectedcells. These studies utilized flow cytometric assessment of labeledcells to provide an objective analysis of phage binding to infectedcells. Two different virus types (HIV and influenza) were used to assessphage that bind TSG101 on the surface of infected cells. Non-infectedcells provided a negative control. These assays identified scFv, encodedin phage, which demonstrated the ability to bind virus-infected cells.FIG. 26.

Those phage encoded scFv candidates that selectively recognized thesurface of virus-infected cells were modified to engineer the scFv intofull length IgG1 antibodies. The resulting antibodies were then screenedusing the same criteria as indicated above. The antibodies derived fromthe CB8 phage provide a specific example.

As a further indication of specificity, all of the resulting antibodiesor phage candidates were evaluated for their binding to differentregions of TSG101 (using the ELISA techniques detailed above). Most ofthe candidates, including CB8, recognized the C-terminal region ofTSG101 while at least one candidate recognized an epitope in the UEVregion.

CB8 antibodies were able to recognize the surface of virus infectedcells. For example, CB8 antibodies selectively recognized human Hep2cells that had been infected with influenza virus. Likewise, theseantibodies were broad-spectrum in their recognition of cells infected bydifferent viruses as the same antibodies recognized human MT4 Tlymphocyte cells that had been infected with HIV. FIG. 27. Similarly,these antibodies selectively recognized non-human cell models that hadbeen infected with virus, including canine MDCK cells infected withinfluenza and primate Vero cells that had been infected with Ebolavirus.

Functional Genetics, Inc. maintains pure stocks of antibodies C, D1, E,3G1 and CB8 as described above, coded to those designations. A depositsof antibodies CB8 has been made at the American Type Culture Collection(ATCC), P.O. Box 1549, Manassas, VA 20108 under Budapest TreatyCondittions as Deposit PTA-9611. These antibodies are available fromFunctional Genetics, Inc. under the terms set forth in the BudapestTreaty for biologic materials including antibodies that are the subjectof a pending patent application.

In addition, techniques developed for the production of “chimericantibodies” (Morrison, et al., 1984, Proc. Natl. Acad. Sci., 81,6851-6855; Neuberger, et al., 1984, Nature 312, 604-608; Takeda, et al.,1985, Nature, 314, 452-454) by splicing the genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. (See, e.g., Cabilly, et al., U.S. Pat. No. 4,816,567; and Boss,et al., U.S. Pat. No. 4,816,397, which are incorporated herein byreference in their entirety.)

Humanized antibodies are antibody molecules from non-human specieshaving one or more complementarity determining regions (CDRs) from thenon-human species and a framework region from a human immunoglobulinmolecule. (see e.g., U.S. Pat. No. 5,585,089, which is incorporatedherein by reference in its entirety.) Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in PCT PublicationNo. WO 87/02671; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication No. WO 86/01533; U.S. Pat. Nos. 4,816,567 and 5,225,539;European Patent Application 125,023; Better, et al., 1988, Science240:1041-1043; Liu, et al., 1987, Proc. Natl. Acad. Sci. USA84:3439-3443; Liu, et al., 1987, J. Immunol. 139:3521-3526; Sun, et al.,1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura, et al., 1987,Canc. Res. 47:999-1005; Wood, et al., 1985, Nature 314, 446-449; Shaw,et al., 1988, J. Natl. Cancer Inst. 80, 1553-1559; Morrison 1985,Science 229:1202-1207; Oi, et al., 1986, Bio/Techniques 4, 214; Jones,et al., 1986, Nature 321, 552-525; Verhoeyan, et al., 1988, Science 239,1534; and Beidler, et al., 1988, J. Immunol. 141, 4053-4060.

Complementarity determining region (CDR) grafting is another method ofhumanizing antibodies. It involves reshaping murine antibodies in orderto transfer full antigen specificity and binding affinity to a humanframework (Winter, et al. U.S. Pat. No. 5,225,539). CDR-graftedantibodies have been successfully constructed against various antigens,for example, antibodies against IL-2 receptor as described in Queen, etal., 1989 (Proc. Natl. Acad. Sci. USA 86, 10029); antibodies againstcell surface receptors-CAMPATH as described in Riechmann, et al. (1988,Nature, 332, 323; antibodies against hepatitis B in Cole, et al. (1991,Proc. Natl. Acad. Sci. USA 88, 2869); as well as against viralantigens-respiratory syncitial virus in Tempest, et al. (1991,Bio-Technology 9, 267). CDR-grafted antibodies are generated in whichthe CDRs of the murine monoclonal antibody are grafted into a humanantibody. Following grafting, most antibodies benefit from additionalamino acid changes in the framework region to maintain affinity,presumably because framework residues are necessary to maintain CDRconformation, and some framework residues have been demonstrated to bepart of the antigen binding site. However, in order to preserve theframework region so as not to introduce any antigenic site, the sequenceis compared with established germline sequences followed by computermodeling.

Completely human antibodies, such as CB8, are particularly desirable fortherapeutic treatment of human patients. Such antibodies can be producedusing transgenic mice which are incapable of expressing endogenousimmunoglobulin heavy and light chain genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a TSG101 protein.

Monoclonal antibodies directed against a TSG101 protein can be obtainedusing conventional hybridoma technology. The human immunoglobulintransgenes harbored by the transgenic mice rearrange during B celldifferentiation, and subsequently undergo class switching and somaticmutation. Thus, using such a technique, it is possible to producetherapeutically useful IgG, IgA and IgE antibodies. For an overview ofthis technology for producing human antibodies, see Lonberg and Huszar(1995, Int. Rev. Immunol. 13, 65-93). For a detailed discussion of thistechnology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see e.g., U.S.Pat. Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806. Inaddition, companies such as Abgenix, Inc. (Freemont, Calif., see, forexample, U.S. Pat. No. 5,985,615) and Medarex, Inc. (Princeton, N.J.),can be engaged to provide human antibodies directed against a TSG101protein or a fragment thereof using technology similar to that describedabove.

Completely human antibodies which recognize and bind a selected epitopecan be generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers, et al., 1994, Biotechnology 12,899-903).

A pre-existing anti-TSG101 antibody can be used to isolate additionalantigens of the pathogen by standard techniques, such as affinitychromatography or immunoprecipitation for use as immunogens. Moreover,such an antibody can be used to detect the protein (e.g., in a cellularlysate or cell supernatant) in order to evaluate the abundance andpattern of expression of TSG101 protein. Detection can be facilitated bycoupling the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude 125I, 131I, 35S or 3H.

Production of Polyclonal Anti-TSG101 Antibodies

The anti-TSG101 antibodies can be produced by immunization of a suitableanimal, such as but are not limited to mouse, rabbit, and horse.

An immunogenic preparation comprising a TSG101 protein or a fragmentthereof are used to prepare antibodies by immunizing a suitable subject(e.g., rabbit, goat, mouse or other mammal). An appropriate immunogenicpreparation can contain, for example, recombinantly expressed orchemically synthesized TSG101 peptide or polypeptide. The preparationcan further include an adjuvant, such as Freund's complete or incompleteadjuvant, or similar immunostimulatory agent.

A fragment of a TSG101 protein suitable for use as an immunogencomprises at least a portion of the TSG 101 protein that is 8 aminoacids, more preferably 10 amino acids and more preferably still, 15amino acids long.

The invention also provides chimeric or fusion TSG101 polypeptides foruse as immunogens. As used herein, a “chimeric” or “fusion” TSG101polypeptides comprises all or part of a TSG101 polypeptide operablylinked to a heterologous polypeptide. Within the fusion TSG101polypeptide, the term “operably linked” is intended to indicate that theTSG101 polypeptide and the heterologous polypeptide are fused in-frameto each other. The heterologous polypeptide can be fused to theN-terminus or C-terminus of the TSG101 polypeptide.

One useful fusion TSG101 polypeptide is a GST fusion TSG101 polypeptidein which the TSG101 polypeptide is fused to the C-terminus of GSTsequences. Such fusion TSG101 polypeptides can facilitate thepurification of a recombinant TSG101 polypeptide.

In another embodiment, the fusion TSG101 polypeptide contains aheterologous signal sequence at its N-terminus so that the TSG101polypeptide can be secreted and purified to high homogeneity in order toproduce high affinity antibodies. For example, the native signalsequence of an immunogen can be removed and replaced with a signalsequence from another protein. For example, the gp67 secretory sequenceof the baculovirus envelope protein can be used as a heterologous signalsequence (Current Protocols in Molecular Biology, Ausubel, et al., eds.,John Wiley & Sons, 1992). Other examples of eukaryotic heterologoussignal sequences include the secretory sequences of melittin and humanplacental alkaline phosphatase (Stratagene; La Jolla, Calif.). In yetanother example, useful prokaryotic heterologous signal sequencesinclude the phoA secretory signal and the protein A secretory signal(Pharmacia Biotech; Piscataway, N.J.).

In yet another embodiment, the fusion TSG101 polypeptide is animmunoglobulin fusion protein in which all or part of a TSG101polypetide is fused to sequences derived from a member of theimmunoglobulin protein family. The immunoglobulin fusion proteins can beused as immunogens to produce antibodies directed against theTSG101polypetide in a subject.

Chimeric and fusion TSG101 polypeptide can be produced by standardrecombinant DNA techniques. In one embodiment, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (e.g.,Ausubel, et al., supra). Moreover, many expression vectors arecommercially available that already encode a fusion domain (e.g., a GSTpolypeptide). A nucleic acid encoding an immunogen can be cloned intosuch an expression vector such that the fusion domain is linked in-frameto the polypeptide.

The TSG101 immunogenic preparation is then used to immunize a suitableanimal. Preferably, the animal is a specialized transgenic animal thatcan secret human antibody. Non-limiting examples include transgenicmouse strains which can be used to produce a polyclonal population ofantibodies directed to a specific pathogen (Fishwild, et al., 1996,Nature Biotechnology 14, 845-851; Mendez, et al., 1997, Nature Genetics15, 146-156). In one embodiment of the invention, transgenic mice thatharbor the unrearranged human immunoglobulin genes are immunized withthe target immunogens. After a vigorous immune response against theimmunogenic preparation has been elicited in the mice, the blood of themice are collected and a purified preparation of human IgG molecules canbe produced from the plasma or serum. Any method known in the art can beused to obtain the purified preparation of human IgG molecules,including but is not limited to affinity column chromatography usinganti-human IgG antibodies bound to a suitable column matrix. Anti-humanIgG antibodies can be obtained from any sources known in the art, e.g.,from commercial sources such as Dako Corporation and ICN. Thepreparation of IgG molecules produced comprises a polyclonal populationof IgG molecules that bind to the immunogen or immunogens at differentdegree of affinity. Preferably, a substantial fraction of thepreparation are IgG molecules specific to the immunogen or immunogens.Although polyclonal preparations of IgG molecules are described, it isunderstood that polyclonal preparations comprising any one type or anycombination of different types of immunoglobulin molecules are alsoenvisioned and are intended to be within the scope of the presentinvention.

A population of antibodies directed to a TSG101 protein can be producedfrom a phage display library. Polyclonal antibodies can be obtained byaffinity screening of a phage display library having a sufficientlylarge and diverse population of specificities with a TSG101 protein or afragment thereof. Examples of methods and reagents particularly amenablefor use in generating and screening antibody display library can befound in, for example, U.S. Pat. Nos. 5,223,409 and 5,514,548; PCTPublication No. WO 92/18619; PCT Publication No. WO 91/17271; PCTPublication No. WO 92/20791; PCT Publication No. WO 92/15679; PCTPublication No. WO 93/01288; PCT Publication No. WO 92/01047; PCTPublication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs, etal., 1991, Biotechnology 9, 1370-1372; Hay, et al., 1992, Hum. Antibod.Hybridomas 3, 81-85; Huse, et al., 1989, Science 246, 1275-1281;Griffiths, et al., 1993, EMBO J. 12, 725-734. A phage display librarypermits selection of desired antibody or antibodies from a very largepopulation of specificities. An additional advantage of a phage displaylibrary is that the nucleic acids encoding the selected antibodies canbe obtained conveniently, thereby facilitating subsequent constructionof expression vectors.

In other preferred embodiments, the population of antibodies directed toa TSG101 protein or a fragment thereof is produced by a method using thewhole collection of selected displayed antibodies without clonalisolation of individual members as described in U.S. Pat. No. 6,057,098,which is incorporated by reference herein in its entirety. Polyclonalantibodies are obtained by affinity screening of a phage display libraryhaving a sufficiently large repertoire of specificities with, e.g., anantigenic molecule having multiple epitopes, preferably after enrichmentof displayed library members that display multiple antibodies. Thenucleic acids encoding the selected display antibodies are excised andamplified using suitable PCR primers. The nucleic acids can be purifiedby gel electrophoresis such that the full length nucleic acids areisolated. Each of the nucleic acids is then inserted into a suitableexpression vector such that a population of expression vectors havingdifferent inserts is obtained. The population of expression vectors isthen expressed in a suitable host.

Identifying Anti-TSG101 Antibodies that Inhibit Viral Production

The invention provides a method for identifying anti-TSG101 antibodiesthat can be used to inhibit or reduce viral budding. In one embodiment,the invention provides a method for determining the effect of anti-TSG101 antibodies on viral infections using a retroviral infection assay. Amurine leukemia virus (MLV) derived vector which contains an E. colilacZ gene expressed from the long terminal repeat (LTR) promoter(pBMN-Z-INeo) is transfected into an amphotropic murine leukemiaretroviral packaging cell line derived from 293 cells (Phoenix A, ATCC).Retroviruses produced by the Phoenix A helper cells are collected andused to infect a mouse N2A cells (ATCC). Anti-TSG101 antibodies areadded to 293 helper cells 24 hours after the transfection of the MLVvector. The effectiveness of TSG 101 antibodies on viral production isdetermined by the efficiency of viral supernatant to infect the targetcells (N2A). The infection of N2A cells is then determined by cellularstaining of (3-galactosidase activity (positive cells are stained blue,shown as dark spots in FIG. 2).

Typically, phoenix A cells are seeded on poly-D-lysine coated 6-wellplate a day before transfection. Four microgram of pBMN-Z-1-Neo is thentransfected into each well in the presence of 12 ul of Lipofectamine2000 (Invitrogen). Twenty-four hours post-transfection, media arereplaced with 1 ml/well of fresh media containing trichostatin A (3 uM)and 5 or 10 ug of proper anti-TSG 101 antibodies. 24 to 48 hours later,viral supernatants are collected, filtered with 0.2 um filters, and 1 mlof viral supernatant is mixed with 1 ml of fresh media containingpolybrene (10 ug/ml), and then is used to infect one well of N2a cells.48 hours post-infection, N2a cells are fixed and stained with X-Gal asdescribed in the 13-Gal staining kit (Invitrogen). Results aredocumented by digital images. Preferably, anti-TSG101 antibodies thatreduce viral production by at least 10%, 20%, 50%, 70% or 90% areidentified.

In the following exemplary experiments, the two anti-TSG101 antibodies“C” and “E” are tested for their effect on viral infection. Rabbit IgGis used as non-specific antibody control. More than 10 independentexperiments are performed, and representative results are shown in FIG.2. Phoenix helper cells without treatment of antibody (positive control)showed efficient production of retroviruses, and infection of N2A targetcells (left top panel); Rabbit IgG had no effect (left middle panel).The anti-TSG101 antibody “C” reduced viral production by about 20%-60%(left bottom panel). The anti-TSG101 antibody “E” reduced viralproduction by about 50-70% (right top panel). A mixture ofanti-C-terminal and anti-N-terminal antibodies give similar results asthe anti-C terminal antibody alone (Right middle panel). N2a cells thatare not infected by viruses only showed minimal background staining(right bottom panel).

In another embodiment, anti-TSG101 antibodies that can be used toinhibit or reduce viral budding are identified based on their binding tocell surface TSG101, e.g., in a human CD4+ human T cell line H9transfected with HIV (designated as H9ΔBg1). H9ΔBg1 cells are human CD4+T lymphocytes transfected with an envelop-defective HIV construct(deletion of a Bg1 II fragment of HIV genome). The stably transfectedH9ABg1 cells produce a non-infectious form of HIV due to the defectiveHIV envelop, hence cannot infect other H9ΔBg1 cells in the culture. Inone embodiment, the untransfected H9 cells are used as control.Anti-TSG111 antibodies that bind to H9ΔBg1 but not the untransfected H9cells are identified as the antibodies that can be used to inhibit orreduce viral budding.

In a preferred embodiment, binding of an anti-TSG101 antibody to cellsurface TSG101 in HIV producing cells (e.g., H9ΔBg1) and control H9cells is identified by Fluorescence Activated Cell Sorting (FACS). Inone embodiment, both H9ΔBg1 and H9 cells are fixed, incubated withanti-TSG101 antibodies, and then stained with a fluorescence labeledsecondary antibody. The immuno-stained cells are then analyzed by FACS.

In another embodiment, a HIV-1 viral production assay is used to furtherexamine the inhibitory effect of TSG101 on retroviral production. TheHIV-1 vector pNL4-3 is transfected into 293T cells. 24 hours aftertransfaction, an anti-TSG101 antibody and, optionally, a non-specificcontrol antibody are added respectively into the cell cultures. Afteradditional 24 hours incubation, cell lysates are extracted, cell culturesupernatants are collected and HIV-1 virions are purified, e.g., bysucrose gradients. Both cell lysates and purified virions are analyzedby Western blot using, e.g., anti-HIV-1 antibodies such as anti-p55and/or anti-p24 antibodies. Anti-TSG101 antibody that exhibitsignificant inhibition of HIV-1 virion release (e.g., more than 40%,50%, 60%, 70%, or 80% inhibition by density tracing of the Westernblots) can be identified.

In still another embodiment, the effect of an anti-TSG101 antibody onHIV release is evaluated using a HIV release assay based on H9ΔBg1cells. HIV release from H9ΔBg1 cells can be directly measured by HIV p24ELISA of cell culture supernatant. In one embodiment, a plurality ofdifferent concentrations of an anti-TSG101 antibody is incubatedrespectively with H9ΔBg1 cells. In one embodiment, a control antibody(e.g., rabbit IgG at the same concentrations) is also incubatedrespectively with H9ΔBg1 cells. 48 hours after antibody addition,culture supernatants are collected for HIV p24 ELISA. Effect of theanti-TSG101 antibody for inhibition of viral release is then determinedby comparing data of the anti-TSG101 antibody with the data of thecorresponding control antibody.

In still another embodiment, the effect of an anti-TSG101 antibody onHIV infectivity following viral release is determined. In oneembodiment, HIV supernatants from Jurkat cells are used to infect MAGIcells in the presence of an anti-TSG101 antibody. Rabbit IgG can be usedas controls. The anti-TSG101 antibody's effect on HIV infectivity isdetermined by comparing with the control.

Uses of Anti-TSG101 Antibodies for Treatment of Viral Infections

TSG101 antibodies are effective in inhibiting viral production. Theinvention therefore provides a method of treating viral infections,including HIV infection, using TSG101 antibodies, e.g., anti-C-terminalTSG101 antibodies.

Viral Infections

Diseases or disorders that can be treated or prevented by the use of ananti-TSG101 antibody of the present invention include, but are notlimited to, those caused by a ritrovirus, rhabdovirus, or filovirus,hepatitis type A, hepatitis type B, hepatitis type C, influenza,varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplextype II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsachie virus,mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II), any picornaviridae, enteroviruses, caliciviridae, anyof the Norwalk group of viruses, togaviruses, alphaviruses,flaviviruses, such as Dengue virus, coronaviruses, rabies virus, Marburgviruses, Ebola viruses, parainfluenza virus, orthomyxoviruses,bunyaviruses, arenaviruses, reoviruses, rotaviruses, orbiviruses, humanT cell leukemia virus type I, human T cell leukemia virus type II,simian immunodeficiency virus, lentiviruses, polyomaviruses,parvoviruses, Epstein-Barr virus, human herpesvirus-6, cercopithecineherpes virus 1 (B virus), and poxviruses.

Additional diseases or disorders that can be treated or prevented by theuse of an anti-TSG101 antibody of the present invention include, but arenot limited to, those caused by influenza virus, human respiratorysyncytial virus, pseudorabies virus, pseudorabies virus II, swinerotavirus, swine parvovirus, bovine viral diarrhea virus, Newcastledisease virus h, swine flu virus, swine flu virus, foot and mouthdisease virus, hog colera virus, swine influenza virus, African swinefever virus, infectious bovine rhinotracheitis virus, infectiouslaryngotracheitis virus, La Crosse virus, neonatal calf diarrhea virus,Venezuelan equine encephalomyelitis virus, punta toro virus, murineleukemia virus, mouse mammary tumor virus, equine influenza virus orequine herpesvirus, bovine respiratory syncytial virus or bovineparainfluenza virus.

Methods of Using Anti-TSG101 Antibodies for Inhibiting Viral Release

In one embodiment, the present invention provides methods of usinganti-TSG101 antibodies, preferably anti-C-terminal TSG101 antibodies, ininhibiting or reducing viral budding, such as HIV-1 budding, frominfected mammalian cells. In the methods of the invention, one or moreanti-TSG101 antibodies are allowed to contact an infected cell. Theanti-TSG101 antibodies binds to the TSG101 protein on the surface of theinfected cell. The binding of the anti-TSG101 antibodies inhibits orreduces the release, or budding, of viral particles from the cell.

In another embodiment, the present invention thus also provides methodsusing anti-TSG101 antibodies, preferably anti-C-terminal TSG101antibodies, for treating infection by an enveloped virus, e.g., HIV-1,in a mammal, e.g., a human. In the methods of the invention, one or moreanti-TSG101 antibodies can be administered to an mammal, e.g., a human,infected by the virus. After administration, the anti-TSG101 antibodiesbind to TSG101 protein on the surface of an infected cell and inhibitingviral budding from the infected cell.

In still another embodiment of the invention, the anti-TSG101antibodies, preferably anti-C-terminal TSG101 antibodies are used inconjunction with one or more other therapeutic anti-viral drugs. In suchcombined therapies, the anti-TSG101 antibodies can be administeredbefore, at the same time, or after the administration of the therapeuticdrugs. The time intervals between the administration of the anti-TSG101antibodies and the therapeutic drugs can be determined by routineexperiments that are familiar to one skilled in the art.

In still another embodiment, the present invention provides a method fortreatment of viral infection using an anti-TSG101 antibody, e.g., ananti-C-terminal TSG101 antibody, that belongs to an isotype that iscapable of mediating the lysis of infected cells to which theanti-TSG101 antibody is bound. In a preferred embodiment, theanti-TSG101 antibody belongs to an isotype that binds a growth factorreceptor and activates serum complement and/or mediates antibodydependent cellular cytotoxicity (ADCC) by activating effector cells,e.g., macrophages. In another preferred embodiment, the isotype is IgG1,IgG2a, IgG3 or IgM.

In this respect, as a further indication of activity of anantibody-based drug, the antibody candidates have been screened fortheir ability to selectively kill virus-infected cells. For this, targetcells were labeled with a vital dye. These cells were then infected withvirus and subjected to ADCC assays, using NK cells derived from normaldonors as effector cells. These studies demonstrated robust andselective killing of virally infected cells by CB8 antibodies. FIG. 29.These antibodies were quite potent, as evidenced by the fact that atleast 70% of infected cells could be killed at antibody concentrationsof 125 ng/mL of CB8 antibody and at relatively low effector:targetratios (3:1). This remarkable selective killing of virally-infectedcells has been reproduced with multiple and different target cells types(Hep2, MT4, MDCK), multiple and different donors (of NK cells) andmultiple and different viruses (HIV, Influenza).

Using other TSG101 antibodies, we have demonstrated that antibodytargeting of TSG101 can directly inhibit viral infection independent ofinnate host defense mechanisms (ADCC or CDC). This finding is consistentwith evidence that TSG101 interactions with viral late domain proteinsare essential for the propagation of many different viruses (see FIG.30). These results lead us to anticipate that many or all of theantibody candidates listed above could have the ability to directlyinhibit viral infection by blocking critical interactions.

The dosage of the anti-TSG101 antibodies can be determined by routineexperiments that are familiar to one skilled in the art. The effects orbenefits of administration of the anti-TSG101 antibodies can beevaluated by any methods known in the art. The compounds could be usedalone or in combination with the current standards of care for any ofthe viruses indicated above. In general, although other modes ofadministration are contemplated, IV or IM injection, or sustained IVadministration, are preferred routes. Dosages will vary from mammal tomammal and virus to virus. Those of skill in the art are well equippedby conventional protocols, given the identification of targets andcompounds herein, to identify specific dosages for specific mammals,specific viruses and specific modes of administration

Methods of Using Anti-TSG101 Antibodies for Delivering Therapeuticand/or Diagnostic Agents

The invention provides methods and compositions for using anti-TSG101antibodies for delivering therapeutic and/or diagnostic agents to viralinfected cells.

Infected cells can be targeted and killed using anti-TSG101antibody-drug conjugates. For example, an anti-TSG101 antibody may beconjugated to a therapeutic moiety such as a cytotoxin, e.g., acytostatic or cytocidal agent, or a radioactive metal ion. Antibody-drugconjugates can be prepared by method known in the art (see, e.g.,Immunoconjugates, Vogel, ed. 1987; Targeted Drugs, Goldberg, ed. 1983;Antibody Mediated Delivery Systems, Rodwell, ed. 1988). Therapeuticdrugs, such as but are not limited to, paclitaxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof, can be conjugated to anti-TSG101 antibodies of theinvention. Other therapeutic agents that can be conjugated toanti-TSG101 antibodies of the invention include, but are not limited to,antimetabolites, e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine; alkylating agents, e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin; anthracyclines, e.g., daunorubicin (daunomycin) anddoxorubicin; antibiotics, e.g., dactinomycin (actinomycin), bleomycin,mithramycin, anthramycin (AMC); and anti-mitotic agents, e.g.,vincristine and vinblastine. The therapeutic agents that can beconjugated to anti-TSG101 antibodies of the invention may also be aprotein or polypeptide possessing a desired biological activity. Suchproteins may include, for example, a toxin such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin.

The drug molecules can be linked to the anti-TSG101 antibody via alinker. Any suitable linker can be used for the preparation of suchconjugates. In some embodiments, the linker can be a linker that allowsthe drug molecules to be released from the conjugates in unmodified format the target site.

The antibodies can also be used diagnostically to, for example, monitorthe progression of a viral infection as part of a clinical testingprocedure to, e.g., determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive materials, positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions. See generally U.S. Pat. No. 4,741,900 for metalions which can be conjugated to antibodies for use as diagnosticsaccording to the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include fluorescent proteins, e.g., greenfluorescent protein (GFP), umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In ¹⁷⁷Lu, ⁹⁰Y or ⁹⁹Tc.

Techniques for conjugating therapeutic moieties to antibodies are wellknown, see, e.g., Amon, et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld, et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom, et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson, et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84:Biological And Clinical Applications, Pinchera, et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin, et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe, et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62, 119-58 (1982); each of which is incorporated hereinby reference.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference.

Detection of Viral Infected Cells

Antibodies or labeled antibodies directed against a Tsg101 protein,e.g., an N-terminal region or a C-terminal region of a TSG101 protein,may also be used as diagnostics and prognostics of viral infection,e.g., by detecting the presence of TSG101 protein on cell surface. Suchdiagnostic methods, may also be used to detect abnormalities in thelevel of Tsg101 gene expression, or abnormalities in the structureand/or temporal, tissue, cellular, or subcellular location of a Tsg101protein.

The tissue or cell type to be analyzed may include those which areknown, or suspected, to be infected by a virus. The protein isolationmethods employed herein may, for example, be such as those described inHarlow and Lane (Harlow, E. and Lane, D., 1988, “Antibodies: ALaboratory Manual”, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.), which is incorporated herein by reference in itsentirety. The isolated cells can be derived from cell culture or from apatient. The analysis of cell taken from culture may be a necessary stepin the assessment of cells to be used as part of a cell-based genetherapy technique or, alternatively, to test the effect of compounds onthe expression of the TSG101 gene.

Preferred diagnostic methods for the detection of TSG101 fragments orconserved variants or peptide fragments thereof, may involve, forexample, immunoassays wherein the TSG101 fragments or conserved variantsor peptide fragments are detected by their interaction with ananti-TSG101 fragment-specific antibody.

For example, antibodies, or fragments of antibodies, such as thosedescribed above useful in the present invention may be used toquantitatively or qualitatively detect infected cells by the presence ofTSG101 fragments or conserved variants or peptide fragments thereof ontheir surfaces. This can be accomplished, for example, byimmunofluorescence techniques employing a fluorescently labeled antibody(see below, this Section) coupled with light microscopic, flowcytometric, or fluorimetric detection. Such techniques are especiallyuseful in viral infection where TSG101 fragments are recruited to thecell surface during the viral budding process.

The antibodies (or fragments thereof) useful in the present inventionmay, additionally, be employed histologically, as in immunofluorescenceor immunoelectron microscopy, for in situ detection of TSG101 fragmentsor conserved variants or peptide fragments thereof. In situ detectionmay be accomplished by removing a histological specimen from a patient,and applying thereto a labeled antibody of the present invention. Theantibody (or fragment) is preferably applied by overlaying the labeledantibody (or fragment) onto a biological sample. Through the use of sucha procedure, it is possible to determine not only the presence of theTSG101 fragment, or conserved variants or peptide fragments, but alsoits distribution in the examined tissue. Using the present invention,those of ordinary skill will readily perceive that any of a wide varietyof histological methods (such as staining procedures) can be modified inorder to achieve such in situ detection.

Immunoassays for TSG101 fragments or conserved variants or peptidefragments thereof will typically comprise incubating a sample, such as abiological fluid, a tissue extract, freshly harvested cells, or lysatesof cells which have been incubated in cell culture, in the presence of adetectably labeled antibody capable of identifying TSG101 fragments orconserved variants or peptide fragments thereof, and detecting the boundantibody by any of a number of techniques well-known in the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled Tsg101 proteinspecific antibody. The solid phase support may then be washed with thebuffer a second time to remove unbound antibody. The amount of boundlabel on solid support may then be detected by conventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tub, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-TSG101 fragment antibody maybe determined according to well known methods. Those skilled in the artwill be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation.

One of the ways in which the TSG101 gene peptide-specific antibody canbe detectably labeled is by linking the same to an enzyme and use in anenzyme immunoassay (EIA) (Voller, A., “The Enzyme Linked ImmunosorbentAssay (ELISA)”, 1978, DiagnosticHorizons 2:1-7, MicrobiologicalAssociates Quarterly Publication, Walkersville, Md.); Voller, A., etal., 1978, J. Clin. Pathol. 31:507-520; Butler, J. E., 1981, Meth.Enzymol. 73:482-523; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRCPress, Boca Raton, Fla.,; Ishikawa, E., et al., (eds.), 1981, EnzymeImmunoassay, Kgaku Shoin, Tokyo). The enzyme which is bound to theantibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietywhich can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes which can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, betagalactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect TSG101 peptides through theuse of a radioimmunoassay (RIA) (see, for example, Weintraub, B.,Principles of Radioimmunoassays, Seventh Training Course on RadioligandAssay Techniques, The Endocrine Society, March, 1986, which isincorporated by reference herein). The radioactive isotope can bedetected by such means as the use of a gamma counter or a scintillationcounter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

Depletion of Viral Infected Cells In Vitro

The invention provides methods of depleting viral infected cells fromnon infected tissues and/or cells in vitro (or ex vivo). For example,the tissue obtained from a mammal for the in vitro depletion of viralinfected cells from non infected cells can be blood or serum or otherbody fluid. In particular, the invention provides for methods ofdepleting viral infected cells by killing them or by separating themfrom non infected cells. In one embodiment, anti-TSG101 antibodies arecombined, e.g., incubated, in vitro with tissues and/or cells obtainedfrom a mammal, e.g., a human.

In one embodiment, a column containing a TSG101 antibody, e.g., anantibody that binds the N-Terminal or C-terminal region of a TSG101protein, bound to a solid matrix is used to remove viral infected cellsfrom a biological sample, e.g., blood or serum or other body fluid.

The anti-TSG101 antibodies used in the in vitro depletion of viralinfected cells from tissues can be conjugated to detectable labels(e.g., various enzymes, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials) or therapeuticagents (e.g., cytostatic and cytocidal agents), which are disclosed insection Methods of Using Anti-SG101 Antibodies for Inhibiting ViralRelease.

Anti-TSG101 antibodies conjugated to detectable substances can beutilized to sort viral infected cells from non infected cells by methodsknown to those of skill in the art. In one embodiment, viral infectedcells are sorted using a fluorescence activated cell sorter (FACS).Fluorescence activated cell sorting (FACS) is a well-known method forseparating particles, including cells, based on the fluorescentproperties of the particles (Kamarch, 1987, Methods Enzymol.,151:150-165). Laser excitation of fluorescent moieties in the individualparticles results in a small electrical charge allowing electromagneticseparation of positive and negative particles from a mixture.

In one embodiment, cells, e.g., blood cells, obtained a mammal, e.g., ahuman, are incubated with fluorescently labeled TSG101 specificantibodies for a time sufficient to allow the labeled antibodies to bindto the cells. In an alternative embodiment, such cells are incubatedwith TSG101 specific antibodies, the cells are washed, and the cells areincubated with a second labeled antibody that recognizes the TSG101specific antibodies. In accordance with these embodiments, the cells arewashed and processed through the cell sorter, allowing separation ofcells that bind both antibodies to be separated from hybrid cells thatdo not bind both antibodies. FACS sorted particles may be directlydeposited into individual wells of 96-well or 384-well plates tofacilitate separation.

In another embodiment, magnetic beads can be used to separate viralinfected cells from non infected cells. Viral infected cells may besorted using a magnetic activated cell sorting (MACS) technique, amethod for separating particles based on their ability to bind magneticbeads (0.5-100 nm diameter) (Dynal, 1995). A variety of usefulmodifications can be performed on the magnetic microspheres, includingcovalent addition of antibody which immunospecifically recognizesTSG101. A magnetic field is then applied, to physically manipulate theselected beads. The beads are then mixed with the cells to allowbinding. Cells are then passed through a magnetic field to separate outviral infected cells.

Dose of Anti-TSG101 Antibodies

The dose can be determined by a physician upon conducting routine tests.Prior to administration to humans, the efficacy is preferably shown inanimal models. Any animal model for an infectious disease known in theart can be used.

In general, for antibodies, the preferred dosage is 0.1 mg/kg to 100mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibodyis to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usuallyappropriate. Generally, partially human antibodies and fully humanantibodies have a longer half-life within the human body than otherantibodies. Accordingly, lower dosages and less frequent administrationare often possible. Modifications such as lipidation can be used tostabilize antibodies and to enhance uptake and tissue penetration (e.g.,into the brain). A method for lipidation of antibodies is described byCruikshank, et al., 1997, J. Acquired Immune Deficiency Syndromes andHuman Retrovirology 14:193.

As defined herein, a therapeutically effective amount of anti-TSG101antibody (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kgbody weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of an anti-TSG101 antibody can include a singletreatment or, preferably, can include a series of treatments. In apreferred example, a subject is treated with an anti-TSG101 antibody inthe range of between about 0.1 to 20 mg/kg body weight, one time perweek for between about 1 to 10 weeks, preferably between 2 to 8 weeks,more preferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. It will also be appreciated that the effectivedosage of an anti-TSG101 antibody, used for treatment may increase ordecrease over the course of a particular treatment. Changes in dosagemay result and become apparent from the results of diagnostic assays asdescribed herein.

It is understood that appropriate doses of anti-TSG101 antibody agentsdepends upon a number of factors within the ken of the ordinarilyskilled physician, veterinarian, or researcher. The dose(s) of theanti-TSG101 antibody will vary, for example, depending upon theidentity, size, and condition of the subject or sample being treated,further depending upon the route by which the composition is to beadministered, if applicable, and the effect which the practitionerdesires the anti-TSG101 antibody to have upon an infectious agent.

Pharmaceutical Formulation and Administration

The anti-TSG101 antibodies of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise anti-TSG101 antibody and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe anti-TSG101 antibody, use thereof in the compositions iscontemplated. Supplementary anti-TSG101 antibodies can also beincorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Preferred routesof administration include subcutaneous and intravenous. Other examplesof routes of administration include parenteral, intradermal, transdermal(topical), and transmucosal. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposablesyringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat the viscosity is low and the anti-TSG101 antibody is injectable. Itmust be stable under the conditions of manufacture and storage and mustbe preserved against the contaminating action of microorganisms such asbacteria and fungi.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating theanti-TSG101 antibody (e.g., one or more anti-TSG101 antibodies) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theanti-TSG101 antibody into a sterile vehicle which contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

In one embodiment, the anti-TSG101 antibodies are prepared with carriersthat will protect the compound against rapid elimination from the body,such as a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811 which is incorporated herein by reference in its entirety.

It is advantageous to formulate parenteral compositions in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of anti-TSG101 antibody calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the anti-TSG101 antibody and the particulartherapeutic effect to be achieved, and the limitations inherent in theart of compounding such an anti-TSG101 antibody for the treatment ofindividuals.

The pharmaceutical compositions can be included in a kit, in acontainer, pack, or dispenser together with instructions foradministration.

TSG101 Vaccines and DNA Vaccines for Treatment and Prevention of ViralInfection

The invention provides fragments of a TSG101 protein which can be usedas vaccines to generate anti-TSG101 antibodies. The TSG101 proteinfragment or polypeptide can be prepared by standard method known in theart. In one embodiment, the invention provides a fragment of a TSG101protein not comprising the UEV domain of a TSG101 protein. In a specificembodiment, the invention provides a fragment of a human TSG101 protein,or its murine homolog, not comprising the UEV domain. In a preferredembodiment, the invention provides a fragment comprising the C-terminalregion of a TSG101 protein. In another embodiment, the inventionprovides a fragment of a TSG101 protein comprising the coiled-coildomain of a TSG101 protein. In still another embodiment, the inventionprovides a fragment of a TSG101 protein comprising C-terminaldomain of aTSG101 protein as described SEQ ID NO:3. The invention also provides anysequence that is at least 30%, 50%, 70%, 90%, or 95% homologous suchfragments of a TSG101 protein. In some embodiments of the invention, theTSG101 protein fragments or polypeptides are at least 5, 10, 20, 50, 100amino acids in length.

The invention also provides fragment of a TSG101 protein which isfunctionally equivalent to any TSG101 fragment described above. Such anequivalent TSG101 fragment may contain deletions, additions orsubstitutions of amino acid residues within the amino acid sequenceencoded by the TSG101 protein gene sequences encoding the TSG101 proteinbut which result in a silent change, thus producing a functionallyequivalent TSG101 protein fragment. Amino acid substitutions may be madeon the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. For example, nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine; polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine;positively charged (basic) amino acids include arginine, lysine, andhistidine; and negatively charged (acidic) amino acids include asparticacid and glutamic acid. “Functionally equivalent”, as utilized herein,refers to a protein fragment capable of exhibiting a substantiallysimilar in vivo activity as the endogenous TSG101 protein fragment.

The TSG101 peptide fragments of the invention may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing the TSG101 polypeptides and peptides of theinvention by expressing nucleic acid containing TSG101 gene sequencesencoding the TSG101 polypeptide or peptide. Methods which are well knownto those skilled in the art can be used to construct expression vectorscontaining TSG101 polypeptide coding sequences and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. See, for example, thetechniques described in Sambrook et al., 1989, supra, and Ausubel, etal., 1989, supra. Alternatively, RNA capable of encoding TSG101polypeptide sequences maybe chemically synthesized using, for example,synthesizers. See, for example, the techniques described in“Oligonucleotide Synthesis”, 1984, Gait, M. J. ed., IRL Press, Oxford,which is incorporated herein by reference in its entirety.

The TSG101 peptide can be used in combination with a suitable carrierand/or adjuvant, such as Freund's complete or incomplete adjuvant, or asimilar immunostimulatory agent. An oil/surfactant based adjuvantcomprising one or more surfactants combined with one or morenon-metabolizable mineral oil or metabolizable oil, such as theIncomplete Seppic Adjuvant (Seppic, Paris, France), may be used. AnIncomplete Seppic Adjuvant has comparable effect as Incomplete Freund'sAdjuvant for antibody production, but induces lower inflammatoryresponse.

The invention also provides portions of a TSG101 gene for use as DNA orRNA vaccine. The TSG101 gene fragments can also be used for producingany TSG101 protein fragment of the invention described above. In apreferred embodiment, the invention provides a fragment of a TSG101 genecomprising the nucleotide region encoding a fragment not comprising theUEV domain of a TSG101 protein. In a specific embodiment, the fragmentof a TSG101 gene is a fragment of a human TSG101 gene, or its murinehomolog. The invention also provides any sequence that is at least 30%,50%, 70%, 90%, or 95% homologous to such fragments of a TSG101 gene. Insome embodiments of the invention, the fragment of a TSG101 gene is atleast 20, 25, 40, 60, 80, 100, 500, 1000 bases in length. Such sequencesmay be useful for production of TSG101 peptides.

The invention also provides (a) DNA vectors that contain any of theforegoing TSG101 coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingTSG101 coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences; and (c) geneticallyengineered host cells that contain any of the foregoing TSG 101 codingsequences operatively associated with a regulatory element that directsthe expression of the coding sequences in the host cell for use inproducing a TSG101 protein fragment of the invention. As used herein,regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include but are not limited to the cytomegalovirushCMV immediate early gene, the early or late promoters of SV40adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage A, the controlregions of fd coat protein, the promoter for 3-phosphoglycerate kinase,the promoters of acid phosphatase, and the promoters of the yeasta-mating factors.

In another embodiment, the present invention provides a naked DNA or RNAvaccine, and uses thereof. The TSG101 DNA fragment of the presentinvention described above can be administered as a vaccine to inhibitviral disease by eliciting anti-TSG101 antibodies of the invention. TheDNA can be converted to RNA for example by subcloning the DNA into atranscriptional vector, such as pGEM family of plasmid vectors, or undercontrol of a transcriptional. promoter of a virus such as vaccinia, andthe RNA used as a naked RNA vaccine. The naked DNA or RNA vaccine can beinjected alone, or combined with one or more DNA or RNA vaccinesdirected to the virus.

The naked DNA or RNA vaccine of the present invention can beadministered for example intermuscularly, or alternatively, can be usedin nose drops. The DNA or RNA fragment or a portion thereof can beinjected as naked DNA or RNA, as DNA or RNA encapsulated in liposomes,as DNA or RNA entrapped in proteoliposomes containing viral envelopereceptor proteins (Nicolau, C. et al. Proc. Natl. Acad. Sci. U.S.A.1983, 80, 1068; Kanoda, Y., et al. Science 1989, 243, 375; Mannino, R.J., et al. Biotechniques 1988, 6, 682). Alternatively, the DNA can beinjected along with a carrier. A carrier can be a protein or such as acytokine, for example interleukin 2, or a polylysine-glycoproteincarrier (Wu, G. Y. and Wu, C. H., J. Biol. Chem. 1988, 263, 14621), or anonreplicating vector, for example expression vectors containing eitherthe Rous sarcoma virus or cytomegalovirus promoters. Such carrierproteins and vectors and methods for using same are known to a person inthe art (See for example, Acsadi, G., et al. Nature 1991, 352, 815-818).In addition, the DNA or RNA could be coated onto tiny gold beads and thebeads introduced into the skin with, for example, a gene gun (Cohen, J.Science 1993, 259, 1691-1692; Ulmer, J. B., et al. Science 1993, 259,1745-1749).

The invention also provides methods for treating a viral infection,e.g., HIV infection, in an animal by gene therapy. A variety of genetherapy approaches may be used to introduce nucleic acid encoding afragment of the TSG101 protein in vivo into cells so as to produceTSG101 antibodies.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow. For general reviews of the methods of gene therapy, seeGoldspiel, et al., 1993, Clinical Pharmacy 12, 488-505; Wu and Wu, 1991,Biotherapy 3, 87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32,573-596; Mulligan, 1993, Science 260, 926-932; and Morgan and Anderson,1993, Ann. Rev. Biochem. 62, 191-217; May, 1993, TIBTECH 11, 155-215).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel, et al. (eds.), 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, New York; andKriegler, 1990, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, New York.

In a preferred aspect, the therapeutic comprises a TSG101 nucleic acidthat is part of an expression vector that expresses a TSG101 or fragmentor chimeric protein thereof in a suitable host. In particular, such anucleic acid has a promoter operably linked to the TSG101 coding region,said promoter being inducible or constitutive, and, optionally,tissue-specific. In another particular embodiment, a nucleic acidmolecule is used in which the TSG101 coding sequences and any otherdesired sequences are flanked by regions that promote homologousrecombination at a desired site in the genome, thus providing forintrachromosomal expression of the TSG101 nucleic acid (see e.g., Kollerand Smithies, 1989, Proc. Natl. Acad. Sci. USA 86, 8932-8935; Zijlstra,et al., 1989, Nature 342, 435-438).

Delivery of the nucleic acid into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe patient. These two approaches are known, respectively, as in vivo orex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded product. This can beaccomplished by any of numerous methods known in the art, e.g., byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., byinfection using a defective or attenuated retroviral or other viralvector (see U.S. Pat. No. 4,980,286), or by direct injection of nakedDNA, or by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, encapsulation in liposomes, microparticles, ormicrocapsules, or by administering it in linkage to a peptide which isknown to enter the nucleus, by administering it in linkage to a ligandsubject to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J.Biol. Chem. 262:4429-4432) (which can be used to target cell typesspecifically expressing the receptors), etc. In another embodiment, anucleic acid-ligand complex can be formed in which the ligand comprisesa fusogenic viral peptide to disrupt endosomes, allowing the nucleicacid to avoid lysosomal degradation. In yet another embodiment, thenucleic acid can be targeted in vivo for cell specific uptake andexpression, by targeting a specific receptor (see, e.g., PCTPublications WO 92/06180 dated Apr. 16, 1992 (Wu, et al.); WO 92/22635dated Dec. 23, 1992 (Wilson, et al.); WO92/20316 dated Nov. 26, 1992(Findeis, et al.); WO93/14188 dated Jul. 22, 1993 (Clarke, et al.), WO93/20221 dated Oct. 14, 1993 (Young)). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, 1989,Proc. Natl. Acad. Sci. USA 86, 8932-8935; Zijlstra, et al., 1989, Nature342, 435-438).

In a specific embodiment, a viral vector that contains the TSG101nucleic acid is used. For example, a retroviral vector can be used (seeMiller, et al., 1993, Meth. Enzymol. 217:581-599). These retroviralvectors have been modified to delete retroviral sequences that are notnecessary for packaging of the viral genome and integration into hostcell DNA. The TSG101 nucleic acid to be used in gene therapy is clonedinto the vector, which facilitates delivery of the gene into a patient.More detail about retroviral vectors can be found in Boesen, et al.,1994, Biotherapy 6, 291-302, which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Clowes,et al., 1994, J. Clin. Invest. 93, 644-651; Kiem, et al., 1994, Blood83, 1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4,129-141; and Grossman and Wilson, 1993, Curr. Opin. Genet. and Devel. 3,110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson (1993,Current Opinion in Genetics and Development 3, 499-503) present a reviewof adenovirus-based gene therapy. Bout, et al. (1994, Human Gene Therapy5, 3-10) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld, et al., 1991,Science 252, 431-434; Rosenfeld, et al., 1992, Cell 68, 143-155; andMastrangeli, et al., 1993, J. Clin. Invest. 91, 225-234.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh, et al., 1993, Proc. Soc. Exp. Biol. Med. 204, 289-300).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217, 599-618; Cohen, et al., 1993, Meth. Enzymol. 217, 618-644;Cline, 1985, Pharmac. Ther. 29, 69-92) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. In a preferred embodiment, epithelial cellsare injected, e.g., subcutaneously. In another embodiment, recombinantskin cells may be applied as a skin graft onto the patient. Recombinantblood cells (e.g., hematopoietic stem or progenitor cells) arepreferably administered intravenously. The amount of cells envisionedfor use depends on the desired effect, patient state, etc., and can bedetermined by one skilled person in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy, aTSG101 nucleic acid is introduced into the cells such that it isexpressible by the cells or their progeny, and the recombinant cells arethen administered in vivo for therapeutic effect. In a specificembodiment, stem or progenitor cells are used. Any stem and/orprogenitor cells which can be isolated and maintained in vitro canpotentially be used in accordance with this embodiment of the presentinvention. Such stem cells include but are not limited to hematopoieticstem cells (HSC), stem cells of epithelial tissues such as the skin andthe lining of the gut, embryonic heart muscle cells, liver stem cells(PCT Publication WO 94/08598), and neural stem cells (Stemple andAnderson, 1992, Cell 71, 973-985).

Epithelial stem cells (ESCs) or keratinocytes can be obtained fromtissues such as the skin and the lining of the gut by known procedures(Rheinwald, 1980, Meth. Cell Bio. 21A:229). In stratified epithelialtissue such as the skin, renewal occurs by mitosis of stem cells withinthe germinal layer, the layer closest to the basal lamina. Stem cellswithin the lining of the gut provide for a rapid renewal rate of thistissue. ESCs or keratinocytes obtained from the skin or lining of thegut of a patient or donor can be grown in tissue culture (Rheinwald,1980, Meth. Cell Bio. 21A, 229; Pittelkow and Scott, 1986, Mayo ClinicProc. 61, 771). If the ESCs are provided by a donor, a method forsuppression of host versus graft reactivity (e.g., irradiation, drug orantibody administration to promote moderate immunosuppression) can alsobe used.

With respect to hematopoietic stem cells (HSC), any technique whichprovides for the isolation, propagation, and maintenance in vitro of HSCcan be used in this embodiment of the invention. Techniques by whichthis may be accomplished include (a) the isolation and establishment ofHSC cultures from bone marrow cells isolated from the future host, or adonor, or (b) the use of previously established long-term HSC cultures,which may be allogeneic or xenogeneic. Non-autologous HSC are usedpreferably in conjunction with a method of suppressing transplantationimmune reactions of the future host/patient. In a particular embodimentof the present invention, human bone marrow cells can be obtained fromthe posterior iliac crest by needle aspiration (see e.g., Kodo, et al.,1984, J. Clin. Invest. 73, 1377-1384). In a preferred embodiment of thepresent invention, the HSCs can be made highly enriched or insubstantially pure form. This enrichment can be accomplished before,during, or after long-term culturing, and can be done by any techniquesknown in the art. Long-term cultures of bone marrow cells can beestablished and maintained by using, for example, modified Dexter cellculture techniques (Dexter, et al., 1977, J. Cell Physiol. 91, 335) orWitlock-Witte culture techniques (Witlock and Witte, 1982, Proc. Natl.Acad. Sci. USA 79, 3608-3612).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Additional methods that can be adapted for use to deliver a nucleic acidencoding a TSG101 fragment of the invention or functional derivativethereof are described below.

Broad Applicability of TSG101 Antibodies to Treatment of Many Virus andVirus Related Diseases

Using TSG101 antibodies, we have demonstrated that antibody targeting ofTSG101 can directly inhibit viral infection independent of innate hostdefense mechanisms (ADCC or CDC). This finding is consistent withevidence that TSG101 interactions with viral late domain proteins areessential for the propagation of many different viruses. These resultslead us to the conclusion that many or all of the TSG101 antibodycandidates of this invention could have the ability to directly inhibitviral infection by blocking critical interactions.

Based on the known involvement of TSG101 in many different viralinfections, the activity reported herein is anticipated to be relevantto infections and diseases caused by multiple and different viruses. Apartial listing of these viruses can be found in Figure XX and involvesvirtually all known viral Groupings.

Viral Groupings:

-   -   Group I: viruses possess double-stranded DNA and include such        virus families as Herpesviridae (examples like HSV1 (oral        herpes), HSV2 (genital herpes), VZV (chickenpox), EBV        (Epstein-Barr virus), CMV (Cytomegalovirus)), Poxyiridae        (smallpox) and many tailed bacteriophages. The mimivirus was        also placed into this group.    -   Group II: viruses possess single-stranded DNA and include such        virus families as Parvoviridae and the important bacteriophage        M13.

Virion- Type of naked/ Capsid nucleic Virus Family Virus Genus envelopedSymmetry acid 1. Adenoviridae Adenovirus Naked Icosahedral ds 2.Papovaviridae Papillomavirus Naked Icosahedral ds circular 3.Parvoviridae B 19 virus Naked Icosahedral ss 4. Herpesviridae HerpesSimplex Virus, Varicella Enveloped Icosahedral ds zoster virus,Cytomegalovirus, Epstein Barr virus 5. Poxviridae Small pox virus,Vaccinia virus Complex Complex ds coats 6. Hepadnaviridae Hepatitis Bvirus Enveloped Icosahedral ds circular 7. Polyomaviridae Polyoma virus(progressive ? ? ds multifocal leucoencephalopathy)

RNA Viruses

-   -   Group III: viruses possess double-stranded RNA genomes, e.g.        rotavirus. These genomes are always segmented.    -   Group IV: viruses possess positive-sense single-stranded RNA        genomes. Many well known viruses are found in this group,        including the picornaviruses (which is a family of viruses that        includes well-known viruses like Hepatitis A virus,        enteroviruses, rhinoviruses, poliovirus, and foot-and-mouth        virus), SARS virus, hepatitis C virus, yellow fever virus, and        rubella virus.    -   Group V: viruses possess negative-sense single-stranded RNA        genomes. The deadly Ebola and Marburg viruses are well known        members of this group, along with influenza virus, measles,        mumps and rabies.

Virion- Type of naked/ Capsid nucleic Virus Family Virus Generaenveloped Symmetry acid  1. Reoviridae Reovirus, Rotavirus NakedIcosahedral ds  2. Picornaviridae Enterovirus, Rhinovirus, NakedIcosahedral ss Hepatovirus, Cardiovirus, Aphthovirus, Parechovirus,Erbovirus, Kobuvirus, Teschovirus  3. Caliciviridae Norwalk virus,Hepatitis E virus Naked Icosahedral ss  4. Togaviridae Rubella virusEnveloped Icosahedral ss  5. Arenaviridae Lymphocytic choriomeningitisEnveloped Complex ss virus  6. Retroviridae HIV-1, HIV-2, HTLV-IEnveloped Complex ss  7. Flaviviridae Dengue virus, Hepatitis C virus,Enveloped Complex ss Yellow fever virus  8. OrthomyxoviridaeInfluenzavirus A, Influenzavirus B, Enveloped Helical ss InfluenzavirusC, Isavirus, Thogotovirus  9. Paramyxoviridae Measles virus, Mumpsvirus, Enveloped Helical ss Respiratory syncytial virus 10. BunyaviridaeCalifornia encephalitis virus, Enveloped Helical ss Hantavirus 11.Rhabdoviridae Rabies virus Enveloped Helical ss 12. Filoviridae Ebolavirus, Marburg virus Enveloped Helical ss 13. Coronaviridae Corona virusEnveloped Complex ss 14. Astroviridae Astrovirus Naked Icosahedral ss15. Bornaviridae Borna disease virus Enveloped Helical ssReverse Transcribing Viruses

-   -   Group VI: viruses possess single-stranded RNA genomes and        replicate using reverse transcriptase. The retroviruses are        included in this group, of which HIV is a member.    -   Group VII: viruses possess double-stranded DNA genomes and        replicate using reverse transcriptase. The hepatitis B virus can        be found in this group.

We have discovered that many of TSG101 is necessary for the propagationof many different viruses and is highly conserved among mammalian oreukaryotic species. Consequently, these compounds could have applicationfor human or veterinary viral diseases. These viral diseases couldinclude but are not limited to PRRS virus, porcine or bovinecircoviruses, porcine or bovine coronaviruses, porcine or bovine RSV,porcine, bovine or avian influenza, EIAV, bluetongue, or foot and mouthdiseases (FMD) viruses.

Some viruses are causative of more chronic diseases and the morbidity ormortality relates to the presence of virus. These diseases includehepatocellular carcinoma (associated with either HBV or HCV), chronicfatigue syndrome (associated with EBV) and other diseases linked withviral infection.

The compounds above could be used for the treatment or prevention(prophylaxis) of single viral pathogens (e.g., HIV or HBV) orcombinations thereof (HIV and HBV). Likewise, these individual orbroad-spectrum applications could entail any or all of the virus groupsdetailed above.

Another method could be the use of the compounds for certain indicationsassociated with one or more viruses. For example, these antibodies couldbe used for the prevention or treatment of respiratory virus infections,which can be caused by one or more of the pathogens from the groupsidentified above. As shown in FIG. 28, the anti-TSG101 antibodies of theinvention bind to RSV infected cells. The administration of this agentto an individual in need of RSV therapy simultaneously providesprophylaxis against opportunistic influenza, a typical combination ofviral agents. Likewise, these compounds could have application againstone or more blood-borne pathogens (e.g., HIV and/or HBV and HCV).

The compounds could have application for the prevention, treatment ormaintenance of acute or chronic viruses. Acute applications includeshort-term prevention or treatment of viral infection, examples of whichinclude influenza, rotavirus or filovirus infection. Chronicapplications could include recurrent outbreaks, such as is observed withgenital herpes) or infrequent outbreaks (such as those associated withzoster infection during shingles). Likewise, treatment could be intendedover the long term to maintain low levels of viral load for chronicvirus infection (e.g., for HIV, HBV or HCV treatment).

EXAMPLES Example 1 Preparation and Uses of Anti-TSG101 PolyclonalAntibodies

To determine the effect of anti-TSG101 antibodies on viral infections, aretroviral infection assay was developed. A murine leukemia virus (MLV)derived vector which contains an E. coli lacZ gene expressed from thelong terminal repeat (LTR) promoter (pBMN-Z-I-Neo) was transfected intoan amphotropic murine leukemia retroviral packaging cell line derivedfrom 293 cells (Phoenix A, ATCC). Retroviruses produced by the Phoenix Ahelper cells were collected and used to infect a mouse N2A cells (ATCC).Anti-TSG101 antibodies were added to 293 helper cells 24 hours after thetransfection of the MLV vector. The effectiveness of TSG101 antibodieson viral production was determined by the efficiency of viralsupernatant to infect the target cells (N2A). The infection of N2A cellswas then determined by cellular staining of P-galactosidase activity(positive cells were stained blue, showed as dark spots in FIG. 2).

Typically, phoenix A cells were seeded on poly-D-lysine coated 6-wellplate a day before transfection. Four microgram of pBMN-Z-I-Neo was thentransfected into each well in the presence of 12 ul of Lipofectamine2000 (Invitrogen). Twenty-four hours post-transfection, media werereplaced with 1 ml/well of fresh media containing trichostatin A (3 uM)and 5 or 10 ug of proper anti-TSG101 antibodies. 24 to 48 hours later,viral supernatants were collected, filtered with 0.2 urn filters, and 1ml of viral supernatant was mixed with 1 ml of fresh media containingpolybrene (10 ug/ml), and then used to infect one well of N2a cells. 48hours post-infection, N2a cells were fixed and stained with X-Gal asdescribed in the 13-Gal staining kit (Invitrogen). Results weredocumented by digital images.

In the following experiments, two anti-TSG101 antibodies were tested fortheir effect on viral infection, a rabbit antibody against N-terminalTSG101 protein, and a rabbit antibody against C-terminal TSG101 protein.The anti-N terminal TSG101 antibody was raised using a N-terminalfragment of the human TSG101 protein: VRETVNVITLYKDLKPVL (SEQ ID NO:2).The anti-C terminal TSG101 antibody was raised using a C-terminalfragment of the human TSG101 protein: QLRALMQKARKTAGLSDLY (SEQ ID NO:3).Rabbit IgG was used as non-specific antibody control. More than 10independent experiments were performed, and representative results areshown in FIG. 2. Phoenix helper cells without treatment of antibody(positive control) showed efficient production of retroviruses, andinfection of N2A target cells (left top panel); Rabbit IgG had no effect(left middle panel). The rabbit antibody against N-terminal TSG101showed about 20%-60% inhibition (left bottom panel). But the rabbitantibody against C-terminal TSG101 significantly inhibited theproduction of retroviruses, and infection of N2A target cells (50-70%inhibition, right top panel). A mixture of the anti-C terminal andanti-N terminal antibodies gave similar results as the anti-C terminalantibody alone (Right middle panel). N2a cells that were not infected byviruses only showed minimal background staining (right bottom panel).Similar results were also obtained in HIV viral infection assays.

Example 2 TSG101 Localized on Cell Surface During Viral Budding

This example shows that domains of TSG101 are exposed on cell surfaceduring HIV release, and anti-TSG101 antibodies inhibited HIV release andinfection.

TSG101 Localization During Viral Release

To demonstrate TSG101 is actively involved viral release at plasmamembrane, an expression vector of GFP-TSG 101 fusion protein wasconstructed and transfected into Phoenix cells (a retroviral helper cellline developed by Nolan et al of Stanford university) that was activelyproducing M-MuLV viruses. 24 hours after transfection, GFP-TSG101 fusionprotein traffic was observed under confocal microscope (Ultraview,Perkin-Elmer). FIGS. 3A-E show a time course of images of GFP-TSG101protein translocation from cytoplasm onto cell surface, and then“budding” out of the viral producing cells.

Cell Surface Localization of TSG101 During HIV Budding

To determine if TSG101 is also actively involved in HIV budding,anti-TSG101 antibodies were used to directly detect cell surface TSG101in a human CD4+ human T cell line H9 transfected with HIV (designated asH9ΔBg1), and the untransfected H9 cells were used as control. The tworabbit anti-TSG101 polyclonal antibodies, one against N-terminal(designated as anti-TSG101 “C”) and one against C-terminal TSG101(designated as anti-TSG101 “E”), were used for this study. Bothantibodies have been well characterized (Li, et al., 2001, Proc. Natl.Acad. Sci. USA 98(4): 1619-24). Both antibodies specifically detectedcell surface localization of TSG101 only in HIV producing H9ΔBg1 cells,and no cell surface TSG101 was detected in control H9 cells (FIG. 4).Interestingly, anti-TSG101 antibodies detected a “capping” like buddingstructure as observed with anti-HIV antibodies (Lee, et al., 1999, J.Virol. 73, 5654-62).

FACS Profile of Cell Surface Localization of TSG 101 During HIV Budding

Cell surface localization of TSG101 in HIV producing cells (H9ΔBg1) andcontrol H9 cells was then examined by Fluorescence Activated Cell Sorter(FACS). Both H9ΔBg1 and H9 cells were fixed, stained with anti-TSG101antibodies, and detected with a fluorescence labeled secondary antibody.The immuno-stained cells were analyzed on FACS. More than sixindependent experiments showed that about 70-85% H9ΔBg1 cells werestained positive for surface TSG101, while less than about 0.1% H9control cells were stained positive for surface TSG101 (FIG. 5). Theseresults were further confirmed by direct examination of both H9ΔBg1 andH9 control cells under confocal microscope. The small population (lessthan 0.1%) of H9 control cells resulted from weak backgroundfluorescence signals associated with immunostaining procedure afterconfocal microscope analysis. The positive population of H9ΔBg1 cellsshowed bright fluorescence.

Anti-TSG101 Polyclonal Antibody Inhibition of HIV Production inTransfected 293 Cells

A HIV-1 viral production assay was used to further examine theinhibitory effect of TSG101 on retroviral production. The HIV-1 vectorpNL4-3 was transfected into 293T cells. 24 hours after transfaction, twoanti-TSG101 antibodies (10 ug/ml), anti-TSG101 antibody “E” andanti-TSG101 antibody “B”, and a non-specific control antibody (10 ug/ml)were added respectively into the cell cultures. Anti-TSG101 antibody “B”was raise against a murine TSG101 N-terminal fragment and binds poorlyto human TSG101 protein. Anti-TSG101 antibody “B” was used as a control.After an additional 24 hours incubation, cell lysates were extracted,cell culture supernatants were collected and HIV-1 virions were purifiedby sucrose gradients. Both cell lysates and purified virions wereanalyzed by Western blot using two anti-HIV-1 antibodies (anti-p55 andanti-p24). As shown in FIG. 6, anti-TSG101 antibody “E” treatment showedsignificant inhibition of HIV-1 virion release (more than 70% inhibitionby density tracing of the Western blots, Lane 8), while anti-TSG101antibody “B” (Lane 7) and the control antibody (Lane 6) did not showsignificant inhibition of HIV-1 release.

Antibody Inhibition of HIV Release from Human CD4+ T Lymphocytes (H₉₀Bg1Cells)

To specifically examine the effect of an antibody on HIV release, a HIVrelease assay based on H9ΔBg1 cells was developed. H9ΔBg1 cells arehuman CD4+ T lymphocytes transfected with an envelop-defective HIVconstruct (deletion of a Bg1 II fragment of HIV genome). The stablytransfected H9ΔBg1 cells produce a non-infectious form of HIV (due tothe defective HIV envelop, hence cannot infect other H9ΔBg1 cells in theculture), HIV release from H9ΔBg1 cells can be directly measured by HIVp24 ELISA of cell culture supernatant. Several concentrations of TSG101antibody “E” and control antibody (rabbit IgG at the sameconcentrations) were used to incubate with H9ΔBg1 cells. 48 hours afterantibody addition, culture supernatants were collected for HIV p24ELISA.

Significant antibody inhibition of viral release was observed at 80ug/ml (FIG. 7).

Inhibition of HIV Infectivity by Polyclonal Anti-TSG101 Antibody

To determine if anti-TSG 101 antibody has additional effect on HIVinfectivity following viral release, HIV supernatants from Jurkat cellswere used to infect MAGI cells in the presence of anti-TSG101 antibody“E” and rabbit IgG as controls (40 ug/ml). Anti-TSG101 antibody showedsignificant inhibition of HIV infectivity (FIG. 8), suggesting TSG101has a role in HIV maturation and/or infection of target cells followingviral release.

Example 3 Effect of Anti-TSG101 Monoclonal Antibodies on HIV Infection

Production of Anti-TSG101 Monoclonal Antibodies (mabs)

A peptide consisting of the nineteen amino acids at C-terminal of TSG101(qlralmqkarktaglsdly, SEQ ID NO:3) was synthesized, conjugated withkeyhole lympet hemocyanin (KLH) and used to immunize 5 mice. Serasamples from each mouse were tested by ELISA against BSA conjugatedC-terminal TSG101 peptide. The mouse with the highest antibody titer wassacrificed and the spleen was fused with myeloma cells to createhybridoma pools. PE-8 is one of the hybridoma pools that contain twohybridomas that produce mabs recognizing the immunizing peptide (SEQ IDNO:3). Pool PE-8 is also referred to as antibody pool PE-8.

Anti-TSG101 Mabs Inhibit Wild Type and Drug-Resistant HIV

Briefly, wild-type and drug resistant HIV expression vector weretransfected into HEK293 cells. The transfected cells were treated withdifferent concentration of anti-TSG101 antibodies and control antibodies24 hours post transfection. Following 24-48 hours incubation afterantibody treatment, cell culture supernatants were collected and used toinfected an indicator cell line (the MAGI cell). The MAGI cells werelysed 24-48 hour post infection and assayed for luciferase activity. Theinhibition of HIV infectivity was determined by reduction of luciferaseactivity. The control antibodies were: N

19, which is an mAb against N-terminal of TSG101; and F-1, F-15 andF-19, which are mAbs raised against the full-length TSG101.

As shown in FIG. 16, antibody pool PE-8, which targets the 19 amino acidresidues at the C-terminal of TSG101 protein and is in the form ofpurified IgG from ascites, inhibits the infectivity of wide type HIVstrain NL4-3 in MAGI cells. In contrast, monoclonal antibodies N-19 andF-1, both are directed to other regions of the TSG101 protein, showed noinhibitory effect on HIV infectivity.

Subclones of the PE-8 mab pool were isolated. The supernatant washarvested from each hybridoma clone and was tested for its anti-HIVactivities. As shown in FIG. 17, the subclones appeared to fall intoeither a

high inhibition

group (e.g., H88 and G12) or a

low inhibition

group (e.g., H10 and F8), which correlates with the fact that the PE-8pool contains only two mabs. In a representative experiment, the mabproduced by the subclone H88 was isolated in the form of purified IgGfrom ascites and was tested for anti-HIV activity. As shown in FIG. 18,the H88 IgG inhibited HIV infection by more than 50%.

As described in more detail in the Background section of theapplication, current treatment for AIDS utilizes inhibitors for thereverse transcriptase (RT) and protease of HIV. However, despite itsinitial success in reducing viral load in the AIDS patient, thetreatment begins to lose efficacy due to the appearance ofdrug-resistant HIV strains in newly infected individuals.

In order to determine whether TSG101 is actively involved in theinfection process of drug-resistant HIV, dominant negative mutants ofTSG101 were generated. Briefly, a TSG101 dominate negative mutantexpression vector was generated by subcloning TSG101 amino acid residues1-312 (SEQ ID NO:42) into a expression vector (pLL1), which contains aCMV promoter and polyA signal.

The TSG101 dominate negative expression vector or a control vector wasthen cotransfected with expression vector for wild-type or drugresistant HIV into HEK293 cells. The culture supernatant were collected24-48 hours post transfection and were used to infected an indicatorcell line (MAGI cells). The MAGI cells were lysed 24-28 hours postinfection and assayed for luciferase activity. The inhibition of HIVinfectivity was determined by reduction of luciferase activity.

As shown in FIG. 19, the dominant negative mutant of TSG101 completelyinhibited the infectivity of RT inhibitor resistant HIV strain pL10R andprotease inhibitor resistant HIV strain p1617-1. It thus appears thatTSG101 plays an essential role in the infectivity of drug resistant HIVstrains.

As demonstrated in FIGS. 20 and 21, anti-TSG101 mab pool PE-8 alsoshowed significant inhibition on the infectivity of the drug-resistantHIV strains pL10R and p1617-1. In contrast, control mabs F-15 and F-19,both were raised against full-length TSG101, showed no inhibition on theinfectivity of the drug-resistant HIV strains. These results suggestthat the anti-TSG101 mabs may provide a new treatment for AIDS,especially for those patients infected by drug-resistant HIV strains.Because the anti-TSG101 antibody treatment targets a host protein ratherthan a viral protein, it would be effective in inhibiting infection byall HIV variants, so long as the TSG101-related pathway is involved inthe viral infection. Moreover, since the treatment does not place adirect selection pressure on the viruses, it would slow the developmentof resistance.

Anti-TSG101 mab inhibits HIV production in human peripheral bloodmononuclear cells (PBMC)

Antibody Inhibition of HIV Infection of Human Peripheral BloodMononuclear Cells (PBMCs)

To determine the effectiveness of anti-TSGIOI antibody inhibition of HIVinfection, a human PBMC based HIV infection assay (Pilgrim, et al., J.Infect Dis. 176:924-32, 1997 and Zhou, et al., Virol. 71:2512-7, 1997)was used. Leukocytes were obtained by leukapheresis of HIV-seronegativedonors, and PBMCs were isolated by Ficoll-Hypaque gradientcentrifugation. Prior to HIV-1 infection, PBMCs were activated byincubation in interleukin-2 (IL-2) cell culture medium containing 10 μgof phytohemagglutinin (PHA) (PHA-P; Difco Laboratories, Detroit, Mich.)per ml. IL-2 culture medium is RPMI 1640 medium containing 100 U ofpenicillin, 100 μg of streptomycin, 2 mM L-glutamine, 10%heat-inactivated fetal calf serum, and 20 U of recombinant IL-2 (RocheMolecular Biochemicals, Indianapolis, Ind.) per ml. After overnightincubation with PHA, cells were washed and continued in culture withIL-2 for 3 to 5 days. All cell cultures were maintained in 5% CO2incubators at 37° C.

HIV-1 isolates were obtained from the National Institutes of Health(NIH) AIDS Research and Reference Reagent Program, Division of AIDS,National Institute of Allergy and Infectious Diseases, NIH (includingwild-type HIV strain pNL4-3, and drug resistant strains pL10-R andpL1617-1), and were expanded by two or three cycles of growth on PHA-and IL-2-stimulated PBMC. To produce the final virus stock, PBMCs wereexposed to undiluted virus for 2 h at a cell concentration of 10⁷/ml,and IL-2 culture medium was added to bring the cell concentration to10⁶/ml. IL-2 culture medium was exchanged every 2 days, and supernatantswere collected during the peak of HIV p24 expression, usually 5 to 10days after infection. Virus stocks were made cell free by centrifugationat 1,000×g and filtration though a 0.45-μm filter. Virus aliquots werestored in the vapor phase of liquid nitrogen. HIV virus 50% tissueculture infectious doses (TCID₅₀) were determined by a sensitive 14-dayendpoint titration assay using PHA and IL-2-stimulated PBMC aspreviously described (Mascola, et al., J. Infect Dis. 173:340-8 1996).

HIV-1 infection of PHA- and IL-2-stimulated PBMC was performed in96-well round-bottomed culture plates by combining 40 μl of virus stockwith 20 μl of PBMC (1.5×10⁵ cells). The multiplicity of infection (MOI)was optimized for individual experiments. Anti-TSG101 antibodyinhibition assay was performed in a 96-well plate format. In this assay,500 to 1,000 TCID₅₀ of HIV-1 were added to each well, resulting in anMOI of about 0.01. PBMC are incubation with antibody and virus for 24hours to 15 days. This assay allows several rounds of virus replication,and therefore virus growth kinetics (measured as extracellular HIV p24production) is monitored by serial collection of culture supernatantsfrom days 2 to 15. HIV p24 is measured with a commercial ELISA kit(Perkin Elmer). Antibody inhibition of HIV infection was determined bysignificant inhibition of the production of infectious HIV particles inthe cell culture supernatant using a MAGI assay as described by Kimpton,et al. and Wei, et al. (Kimpton, et al., J. Viol 66, 2232, 1992; Wei, etal., Antimicro Agents Chemother, 46, 1896, 2002). Multiple time pointsand duration of antibody incubations will be examined for the optimalinhibition of HIV infection of human PBMCs.

As shown in FIG. 22, anti-TSG101 mab pool PE-8 (marked as C8P in thefigures) significantly inhibits HIV production in human PBMC after 3-dayand 7-day incubation, respectively.

Example 4 Molecular Cloning of Anti-TSG101 MAB Genes

Genes encoding the mabs in the PE-8 antibody pool (containing two mabs)were cloned. Briefly, The variable regions of the mab genes wereamplified by RT-PCR with total RNA from the hybridoma cells, using thedegenerate primers designed from the mab leader sequences and theconstant domains near the variable region of both chains. The variabledomain of the heavy chain for PE-8 pool was amplified with a forwardprimer (5′-actagtcgacatgtacttgggactgagctgtgtat-3′ (SEQ ID NO:4)) and areverse primer (5′-cccaagcttccagggrccarkggataracigrtgg-3′ (SEQ IDNO:5)); while the variable domain of the light chain was amplified withforward primers (a mixture of two degenerate primers5′-actagtcgacatggtrtccwcasctcagttccttg-3′ (SEQ ID NO: 6) and5′-actagtcgacatgakgthcycigctcagytyctirg-3′ (SEQ ID NO: 7)) and a reverseprimer (5′-cccaagcttactggatggtgggaagatgga-3′ (SEQ ID NO: 8)). Thevariable domain of the heavy chain of mab3 G1 was amplified with theforward primers (a mixture of three degenerate primers5′-actagtcgacatgatggtgttaagtcttctgtacct-3′ (SEQ ID NO:9),5′-actagtcgacatgaaatgcagctggrtyatsttctt-3′ (SEQ ID NO:10), and5′-actagtcgacatggrcagrcttacwtyytcattcct-3′ (SEQ ID NO:11)) and thereverse primer, 5′-cccaagcttccagggrccarkggataracigrtgg-3′ (SEQ IDNO:12), while the variable domain of the light chain was amplified withthe forward primers (a mixture of four degenerate primers5′-actagtcgacatgaagttgcctgttaggctgttggtgct-3′ (SEQ ID NO:13),5′-actagtcgacatggatttwcargtgcagattwtcagctt-3′ (SEQ ID NO:14),5′-actagtcgacatggtyctyatvtccttgctgttctgg-3′ (SEQ ID NO:15) and5′-actagtcgacatggtyctyatvttrctgctgctatgg 3′ (SEQ ID NO:16) and a reverseprimer, 5′-cccaagcttactggatggtgggaagatgga-3′ (SEQ ID NO:17). The 450-bpPCR products were purified from the agarose gel and ligated into theTA-cloning vector for sequencing confirmation.

Using the above-described method, the heavy and light chain cDNAsequences of clone D1 (a dominant clone) and clone 3G1 of pool PE-8 weredetermined. The amplified cDNA sequences and the variable region nucleicacid and amino acid sequences are summarized in Table 1.

TABLE 1 Cloned mab sequences Variable region Variable region Cloned cDNAamino acid nucleotide Gene sequence sequence sequence Clone D1 heavychain (V_(H)) SEQ ID NO: 18 SEQ ID NO: 19 SEQ ID NO: 20 clone D1 lightchain (V_(L)) SEQ ID NO: 21 SEQ ID NO: 22 SEQ ID NO: 23 clone 3G1 heavychain (V_(H)) SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 26 Clone 3G1 lightchain (V_(L)) SEQ ID NO: 27 SEQ ID NO: 28 SEQ ID NO: 29

The complementarity determining regions (CDRs) of the mabs are listed inTable 2.

TABLE 2 Amino acid sequences of the CDRs of mabs D1 and 3G1 CDRsAmino Acid Sequences D1 V_(H )chain CDR2 QIRNKPFNYETFYSDSV(SEQ ID NO: 31) D1 V_(H )chain CDR3 SNHRYGV (SEQ ID NO: 32)D1 V_(L )chain CDR2 YSSRLQS (SEQ ID NO: 34) D1 V_(L )chain CDR3 QQSKAL(SEQ ID NO: 35) 3G1 V_(H )chain CDR2 HIWWDNV (SEQ ID NO: 37)3G1 V_(H )chain CDR3 ARIEGVNGNYPYF (SEQ ID NO: 38) 3G1 V_(L )chain CDR2GTSNLAS (SEQ ID NO: 40) 3G1 V_(L )chain CDR3 SSYPLMT (SEQ ID NO: 41)

Example 5 Anti-TSG101 Antibodies Inhibit Release of Ebolavirus

This Example shows that TSG101 interacts with EBOV VP40, that TSG101 isincorporated into EBOV VLPs, and that anti-TSG101 antibody inhibits therelease of EBOV virus-like particles (VLPs).

The only members of the family Filoviridae, EBOV and MARV possess anegative-stranded, non-segmented 19 Kb RNA genome comprising 7 genes:nucleoprotein (NP), viral proteins VP35, VP40, glycoprotein (GP), VP30,VP24, and RNA polymerase (L), encoding for seven proteins in MARV andeight proteins in EBOV. Recent studies provide some insights in thecellular localization and role of VP40, a 326 amino acid matrix (M)protein (Jasenosky, et al., 2001, J. Virol. 75 (11): 5205-14;Kolesnikova, et al., 2002, J. Virol 76 (4): 1825-38). In cells infectedwith either EBOV or MARV, the majority of VP40 is peripherallyassociated with the cytoplasmic face of the plasma membrane viahydrophobic interactions. Significantly, expression of EBOV and MARVVP40 in transfected cells is required for the production of virus-likeparticles (VLPs), non-infectious particles that have some morphologicalproperties similar to authentic viruses. The ability of VP40 to directits own release from infected cells was mapped to a proline-richsequence motif common to other enveloped RNA viruses (Harty, et al.,2000, Proc. Natl. Acad. Sci. USA 97, 13871-6).

Generation of Virus-Like Particles as a Surrogate Model for EbolaAssembly and Release

Several virus-like particles can be generated by mere expression ofviral matrix proteins (Johnson, et al., 2000, Curr. Opin. Struct. Biol.10, 229-235). EBOV and MARV matrix proteins (VP40) have been shown tolocalize to both the plasma membrane and viralinclusion bodies(Kolesnikova, et al., 2002, J. Virol. 76, 1825-38; Martin-Serrano, etal., 2001, Nature Medicine 7, 1313-19), suggesting that VP40 may drivethe assembly and release of mature virions. However, attempts toefficiently generate VLPs by expression of VP40 alone have been largelyunfruitful, marked by inefficient release of amorphous VP40-containingmaterial (Bavari, et al., 2002, J. Exp. Med. 195, 593-602). Inretroviruses, the raft localization of the assembly complex is regulatedby the association of N-terminally acylated Gag proteins (Campbell, etal., 2001, J. Clin. Virol. 22, 217-227), whereas raft targeting offilovirus proteins such as VP40 appear to be mainly regulated by theviral glycoprotein (GP) (Bavari, et al., 2002, J. Exp. Med. 195,593-602). Therefore, it was hypothesized that generation of filovirusVLPs may require coexpression of both GP and VP40. Whether GP and VP40are released into culture supernatants was first examined. In cellsexpressing either GP or VP40 alone both proteins could be detected bothin cells and supernatants (FIG. 9A). Coexpression of both proteins,however, resulted in substantial increase in release from cells (FIG.9A). It was reasoned that if the released GP and VP40 are associated inparticles, VP40 must be co-immunoprecipitated with anti-GP mAb. As shownin FIG. 9B, VP40 was readily detected in anti GP-immunoprecipitates fromthe supernatants of cells transfected with both GP and VP40 of EBOV. NoVP40 was pulled down from the supernatant of cells expressing VP40alone, showing that the co-IP is specific.

Particles Formed by EBOV GP and VP40 Display the MorphologicalCharacteristics of Ebola Virus

The co-IP experiments demonstrated that GP and VP40 released intosupernatant are associated with each other in some form. To determinewhether these complexes representvirus-like particles (VLPs),particulate material from culture supernatants was purified by sucrosegradient ultracentrifugation (Bavari, et al., 2002, J. Exp. Med. 195,593-602) and analyzed using electron microscopy. Interestingly, most ofthe particles obtained from the supernatants of the cells cotransfectedwith GP and VP40 displayed a filamentous morphology strikingly similarto filoviruses (FIGS. 10A and 10B) (Geisbert, et al., 1995, Virus Res.39, 129-150; Murphy, et al., 1978, Ebola and Marburg virus morphologyand taxonomy, 1 edn (Amsterdam, Elsvier)). In contrast, the materialobtained from singly transfected cells only contained small quantitiesof membrane fragments, likely released during cell death. The VLPs havea diameter of 50-70 nm and are 1-2 μm in length (FIG. 10). This issimilar to the length range of Ebola virions found in cell culturesupernatants after in vitro infection (Geisbert, et al., 1995, VirusRes. 39, 129-150). The smaller diameter of VLPs (as compared to 80 nmfor EBOV) may be due to the lack of ribonucleoprotein complex. All typesof morphologies described for filoviruses such as branched, rod-, U- and6-shaped forms (Feldmann, et al., 1996, Adv. Virus Res. 47, 1-52;Geisbert, et al., 1995, Virus Res. 39, 129-150) among these particleswere observed (FIG. 10). In addition, the VLPs were coated with 5-10 nmsurface projections or “spikes” (FIG. 10), characteristic for EBOV(Feldmann, et al., 1996, Adv. Virus Res. 47, 1-52; Geisbert, et al.,1995, Virus Res. 39, 129-150). Immunogold staining of the VLPs withanti-Ebola GP antibodies demonstrated the identity of the spikes on thesurface of the particles as Ebola glycoprotein (FIG. 10B). VLPs forMarburg virus were also generated in a similar manner.

In summary, a surrogate assay for the assembly and release of Ebolavirus that can be performed without the restrictions of biocontainmentlaboratories was established. This assay can be used for initialscreenings of agents that may inhibit Ebola virus budding.

Studies on the Role of TSG101 in Ebola Virus Life Cycle

We performed a series of biochemical studies to examine the involvementof the vaccuolar protein sorting (vps) protein TSG101 interaction withthe late domain of VP40 in EBOV assembly and release. A set of TSG101truncations C-terminally tagged with a Myc epitope were used for thesestudies. 293T cells were transfected with full length TSG101 and mutantstruncated at positions 140, 250 and 300 along with EBOV VP40. Cells werelysed after 48 h and subjected to immunoprecipitation with an anti-Mycantibody. As shown in FIG. 11, VP40 was coprecipitated with all TSG101proteins except for the 1-140 truncated mutant. Lack of association withthis mutant is consistent with the structural data that show thatresidues 141-145 make important contacts with an HIV Gag-derived PTAPpeptide (Pornillos, et al., 2002, Nat. Struct. Biol. 9, 812-7).Interestingly, the association of VP40 with 1-300 mutant of TSG101 wassignificantly stronger than with full length or 1-250 mutant (FIG. 11).The lower association of full length TSG101 can be attributed to thepresence of a PTAP motif in the C-terminal region of this molecule thatmay form an 30 inter- or intramolecular association with the UEV domainof TSG101. The dramatic reduction of interaction resulting from deletionof amino acids 250-300 suggests that residues in this region maycontribute to the binding to viral matrix proteins.

To confirm that TSG101 and VP40 associate directly and through the PTAPmotif, Far Western analysis was performed. Ebola VP40 (1-326) andtruncated (31-326) Ebola VP40 proteins as well as HA-tagged UEV domainof TSG101 were produced in bacteria. These proteins were electrophoresedon 4-20% gradient gel and electroblotted onto nitrocellulose membrane.Following blocking, the blot was incubated with a purified TSG101protein (UEV), washed, and protein protein interaction detected byenhanced chemiluminescence using an anti TSG101 antibody and HRP-labeledgoat anti rabbit as secondary antibody. As shown in FIG. 12, TSG101interacts with the full length Ebola VP40 but not with the truncatedEbola VP40, confirming that the PTAP motif at the N terminus of VP40plays a critical role in VP40 TSG101 (UEV) interaction. An identicalwestern blot developed with Ebola VP40 antibody could detect both thefull length and the truncated VP40 showing the presence of both theproteins on the blot.

Surface Plasmon Resonance Biosensor (SPR) Analysis of the Ebola VP40TSG101 Interaction

A quantitative analysis of Ebola VP40 interacting with TSG101 wascarried out using SPR measurements. A biotinylated peptide(Bio-ILPTAPPEYME, SEQ ID NO:43) containing 11 amino acid residues fromthe N terminus of the Ebola VP40 was immobilized on the streptavidinechip. Purified TSG101 protein that contains only the UEV domain wasinjected at different concentrations (1, 2, 5, 20 uM) serially. As seenin the FIG. 13, an interaction of moderate affinity between the peptideand proteins can be detected. Based on the SPR data we calculated a Kdvalue of ˜2 μM for this interaction.

Incorporation of TSG101 in Ebola VLPs and Virions

To determine whether TSG101 is incorporated in EBOV VLPs, TSG101 (1-312,SEQ ID NO:42) together with GP or GP+VP40 were expressed in 293T cells.VLPs were immunoprecipitated from supernatants using anti GP antibodies.Expression of TSG101(1-312) resulted in a marked increase in VLPrelease, suggesting a positive role for TSG101 in VLP budding (FIG. 14A,Lane 4). In addition, TSG101 (1-312) was coimmunoprecipitated with ananti-GP antibody from the culture supernatants when expressed along withGP and VP40 (FIG. 14A, Lane 4). No TSG101 was found associated with GPwhen expressed in the absence of VP40 (FIG. 14A, lane 3), suggestingthat its association with GP was dependent on formation of VLPs. Similarresults were also obtained with full length TSG101. These data stronglysuggest that TSG101 is incorporated into VLPs and support the hypothesisthat TSG101 plays a role in viral assembly and/or budding. To furthersubstantiate this finding we also analyzed inactivated, band purified,EBOV (iEBOV) for the presence of TSG101. 5 μg iEBOV were analyzed byimmunoblotting for the presence of TSG101. As shown in FIG. 6B, we foundreadily detectable levels of TSG101 in iEBOV, clearly demonstrating theincorporation of TSG101 in Ebola virus.

Effect of Polyclonal Anti-TSG101 Antibodies on Ebola Virus Release

The biochemical and VLP release data suggested that TSG101 is criticalfor the egress of Ebola virus. Therefore, the effects of polyclonalanti-TSG101 antibodies “C” and “E” (that showed inhibitory effect onHIV, see Examples 1 and 2) were tested on the virus production in Helacells infected with Ebola Zaire-95 virus. Monolayers of Hela cells wereincubated with the virus at an MOI of 1 for 50 minutes, washed, and amedium containing an anti-TSG101 antibody or a control rabbit anti mouseantibody were added at 5 μg/ml. After 24 hours the supernatants wereharvested and the released viruses enumerated by plaque assay aspreviously described (Bavari, et al., 2002, J. Exp. Med. 195, 593-602).As shown in FIG. 15, these antibodies partially inhibited the release ofvirions into Hela cell supernatant.

Anti-TSG101 Mabs Protects Mice Against EBOV Challenge

The anti-TSG101 mabs were also tested for their anti-EBOV activity inmice. Briefly, Balb/c mice in groups of 10 were injectedintraperitioneally with either 4 mg of the anti-TSG101 mab pool PE-8(shown as mab C8 in Table 3 and FIG. 23) in the form of purified IgG, 4mg of a control mab, or phosphate buffered saline (PBS). One hour afterthe injection, the mice were challenged with EBOV at a dose of 100pfu/mouse. As shown in Table 3 and FIG. 27, injection of anti-TSG101mabs significantly increased the survival rate of EBOV challenged mice.

TABLE 3 Protection of Anti-TSG101 Mab against EBOV Antibody TreatmentSurvivals/total PBS control i.p. on day 0 1/10 Mab control 4 mg i.p. onday 0 1/10 Mab C8 4 mg i.p. on day 0 5/10

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of the present invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims along with the full scope ofequivalents to which such claims are entitled.

Sequences set forth or recited herein, by SEQ. ID NO. Identification 1.mavsesqlkk mvskykyrdl tvretvnvit lykdlkpvldsyvfndgssr elmnltgtip vpyrgntyni piclwlldtypynppicfvk ptssmtiktg khvdangkiy lpylhewkhpqsdllgliqv mivvfgdepp vfsrpisasy ppyqatgppntsympgmpgg ispypsgypp npsgypgcpy ppggpypattssqypsqppv ttvgpsrdgt isedtirasl isavsdklrwrmkeemdraq aelnalkrte edlkkghqkl eemvtrldqevaevdkniel lkkkdeelss alekmenqse nndideviiptaplykqiln lyaeenaied tifylgealr rgvidldvflkhvrllsrkq fqlralmqka rktaglsdly 2. VRETVNVITLYKDLKPVL 3.QLRALMQKARKTAGLSDLY 4. actagtcgacatgtacttgggactgagctgtgtat 5.cccaagcttccagggrccarkggataracigrtgg 6.actagtcgacatggtrtccwcasctcagttccttg 7.actagtcgacatgakgthcycigctcagytyctirg 8. cccaagcttactggatggtgggaagatgga9. actagtcgacatgatggtgttaagtcttctgtacct 10.actagtcgacatgaaatgcagctggrtyatsttctt 11.actagtcgacatggrcagrcttacwtyytcattcct 12.cccaagcttccagggrccarkggataracigrtgg 13.actagtcgacatgaagttgcctgttaggctgttggtgct 14.actagtcgacatggatttwcargtgcagattwtcagctt 15.actagtcgacatggtyctyatvtccttgctgttctg 16.actagtcgacatggtyctyatvttrctgctgctatgg 17. cccaagcttactggatggtgggaagatgga18. ATGTACTTGGGACTGAGCTGTGTATTCATTGTTTTTCTCTTAAAAGGTGT CCAGTGTGAGGTGAAGCTGGATGAGACTGGAGGAGGCTTGGTGCAACCTGGGAGGCCCAT GAAACTCTCGTGTGTTGCCTCTGGATTCACTTTTAGTGACTACTGGATGAACTGGGTCCG CCAGTCTCCAGAGAAGGGACTGGAGTGGGTAGCGCAAATTAGAAACAAACCGTTTAATTA TGAAACATTTTATTCAGATTCTGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCCAA AAGTAGTGTCTACCTGCAAATGAACAACTTAAGAAATGAGGACATGGGTATCTATTACTG TTCAAATCATAGATATGGGGTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGC AGCCAAAACGACACCCCCATCCGTTTATCCCTTGGTCCCTGGAAGCTTGGG 19.EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPFNYETFYSDSVKGRFTISRDDSKSSVYLQMNNLRNEDMGIYYCSN HRYGVAYWGQGTLVTVSA20. GAGGTGAAGCTGGATGAGACTGGAGGAGGCTTGGTGCAACCTGGGAGGCC CATGAAACTCTCGTGTGTTGCCTCTGGATTCACTTTTAGTGACTACTGGATGAACTGGGTCCG CCAGTCTCCAGAGAAGGGACTGGAGTGGGTAGCGCAAATTAGAAACAAACCGTTTAATTA TGAAACATTTTATTCAGATTCTGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCCAA AAGTAGTGTCTACCTGCAAATGAACAACTTAAGAAATGAGGACATGGGTATCTATTACTG TTCAAATCATAGATATGGGGTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGC A 21.CNGTCTGTTGCTCTGTTTTCAGGTACCAGATGTGATATCCAGATGACACA GACTACAACCTCCCTGTCTGCCTCTCTGGGAGACAGGGTCACCATCAGTTGCAGGGCAAG TCAGGACATTAACCATTATTTAAGCTGGTTTCAGCAGAAACCAGATGGAACTGTTAAACT CCTGATCTTCTACTCATCAAGATTACAGTCAGGTGTCCCGTCAAGGTTCAGTGGCAGTGG GTCTGGAAGAGATTTTTCTCTCACCATTAGGGCCCTGGAACAAGAAGATATTGCCACTTA CTTTTGCCAACAAAGTAAAGCGCTCCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAAT CAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAG 22.DIQMTQTTTSLSASLGDRVTISCRASQDINHYLSWFQQKPDGTVKLLIFYSSRLQSGVPSRFSGSGSGRDFSLTIRALEQEDIATYFCQQSKALPWTFGG GTKLEIKR 23.GATATCCAGATGACACAGACTACAACCTCCCTGTCTGCCTCTCTGGGAGACAGGGTCACCATCAGTTGCAGGGCAAG TCAGGACATTAACCATTATTTAAGCTGGTTTCAGCAGAAACCAGATGGAACTGTTAAACT CCTGATCTTCTACTCATCAAGATTACAGTCAGGTGTCCCGTCAAGGTTCAGTGGCAGTGG GTCTGGAAGAGATTTTTCTCTCACCATTAGGGCCCTGGAACAAGAAGATATTGCCACTTA CTTTTGCCAACAAAGTAAAGCGCTCCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAAT CAAACGG 24.GCCCTTACTAGTCGACATGGGCAGGCTTACTTTCTCATTCCTGCTACTGA TTGTCCCTGCATATGTCCTGTCCCAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGC AGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGATTCTCACTGAGCACTTTTA ATGTGGGTGTAGGCTGGATTCGTCAGCCATCAGGGAAGGGTCTGGAGTGGCTGGCACACA TTTGGTGGGATAATGTCAAGCGCTATAACCCAGCCCTGAAGAGCCGACTGACTATCTCCA AGGATACCTCCAGCAGCCAGGTATTCCTCGACATCGCCAGTGTGGACACTGCAGATACTG CCACATATTTTTGTGCTCGAATAGAGGGGGTGAATGGTAACTACCCCTACTTTTCTTACT GGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCCGTTTATC CCCTGGCCCCTGGAAGCTTGGG 25. QVTLKESGPGILQPSQTLSLTCSFSGFSLSTFNVGVGWIRQPSGKGLEWLAHIWWDNVKRYNPALKSRLTISKDTSSSQVFLDIASVDTADTATYFCARIEGVNGNYPYFSYWGQGTLVTVSA 26.ATATGTCCTGTCCCAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGC AGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGATTCTCACTGAGCACTTTTA ATGTGGGTGTAGGCTGGATTCGTCAGCCATCAGGGAAGGGTCTGGAGTGGCTGGCACACA TTTGGTGGGATAATGTCAAGCGCTATAACCCAGCCCTGAAGAGCCGACTGACTATCTCCA AGGATACCTCCAGCAGCCAGGTATTCCTCGACATCGCCAGTGTGGACACTGCAGATACTG CCACATATTTTTGTGCTCGAATAGAGGGGGTGAATGGTAACTACCCCTACTTTTCTTACT GGGGCCAAGGGACTCTGGTCACTGTCTCTGCA 27.ACTAGTCGACATGGATTTACAGGTGCAGATTATCAGCTTCATGCTAATCA GTGTCACAGTCATATTGTCCAGTGGAGAAATTGTGGTCACCCAGTCTCCGGCACTCATGG CTGCATCTCCAGGGGAGAGGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAATTCCA ACAACTTACACTGGTACCAACAGAAGTCAGAAGCCTCCCCCAAACCCTGGATTTATGGCA CATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTT TTTCTCTCACAGTCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGT GGAGTAGTTACCCACTCATGACGTTCGGTGGGGGCACCAAACTGGAAATCAAGCGGGCTG ATGCTGCACCAACTGTATCCATCTTCCACCATCCAGTAAGCTTGGG 28.EIVVTQSPALMAASPGERVTITCSVSSSINSNNLHWYQQKSEASPKPWIYGTSNLASGVPVRFSGSGSGTSFSLTVSSMEAEDAATYYCQQWSSYPLMTF GGGTKLEIKR 29.CATATTGTCCAGTGGAGAAATTGTGGTCACCCAGTCTCCGGCACTCATGG CTGCATCTCCAGGGGAGAGGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAATTCCA ACAACTTACACTGGTACCAACAGAAGTCAGAAGCCTCCCCCAAACCCTGGATTTATGGCA CATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTT TTTCTCTCACAGTCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGT GGAGTAGTTACCCACTCATGACGTTCGGTGGGGGCACCAAACTGGAAATCAAGCGG 30. DYWMN 31.QIRNKPFNYETFYSDSV 32. SNHRYGV 33. RASQDINHYLS 34. YSSRLQS 35. QQSKAL 36.FNVGVG 37. HIWWDNV 38. ARIEGVNGNYPYF 39. RVTITCSVSSSINSNNLH 40. TSNLAS41. SSYPLMT 42. mavsesqlkk mvskykyrdl tvretvnvit lykdlkpvldsyvfndgssr elmnltgtip vpyrgntyni piclwlldtypynppicfvk ptssmtiktg khvdangkiy lpylhewkhpqsdllgliqv mivvfgdepp vfsrpisasy ppyqatgppntsympgmpgg ispypsgypp npsgypgcpy ppggpypattssqypsqppv ttvgpsrdgt isedtirasl isavsdklrwrmkeemdraq aelnalkrte edlkkghqkl eemvtrldqevaevdkniel lkkkdeelss alekmenqse nn 43. ILPTAPPEYME

1. A pharmaceutical composition for inhibiting viral infection due tohuman immunodeficiency virus, Ebola virus or influenza virus in amammalian subject, comprising: 1) an effective amount of an anti-TSG101antibody CB8, ATCC Deposit PTA-9611 or an antibody comprising the entireset of CDRs of antibody CB8, ATCC Deposit PA-9611; and 2) apharmaceutically acceptable carrier.
 2. A method for inhibiting viralbudding in a mammalian subject, comprising providing to said subject aneffective amount of an anti-TSG101 antibody CB8, ATCC Deposit PTA-9611,or an antibody comprising the entire set of complementarity determiningregions (CDRs) of CB8, ATCC Deposit PTA-9611, wherein said antibodybinds TSG101 present on cells of said mammalian subject which may becomeinfected with said virus and wherein said virus is humanimmunodeficiency virus, ebola virus or influenza virus.
 3. The method ofclaim 1, wherein the antibody is CB8.
 4. The method of claim 1, whereinsaid virus is a human immunodeficiency virus.
 5. The method of claim 1,wherein said mammalian subject is a non-human.
 6. The method of claim 1,wherein said anti-TSG101 antibody is provided to said mammalian subjectby administering said antibody to said mammalian subject.
 7. The methodof claim 1, wherein said virus is Ebola virus.
 8. The method of claim 1,wherein said virus is influenza virus.